Stem cells of the islets of langerhans and their use in treating diabetes mellitus

ABSTRACT

Methods and compositions are described for the treatment of type I insulin-dependent diabetes mellitus and other conditions using newly identified stem cells that are capable of differentiation into a variety of pancreatic islet cells, including insulin-producing beta cells, as well as hepatocytes. Nestin and ABCG2 have been identified as molecular markers for pancreatic stem cells, while cytokeratin-19 serves as a marker for a distinct class of islet ductal cells. Methods are described whereby nestin and/or ABCG2-positive stem cells can be isolated from pancreatic islets and cultured to obtain further stem cells or pseudo-islet like structures. Methods for ex vivo differentiation of the pancreatic stem cells are disclosed. Methods are described whereby pancreatic stem cells can be isolated, expanded, and transplanted into a patient in need thereof, either allogeneically, isogeneically or xenogenically, to provide replacement for lost or damaged insulin-secreting cells or other cells.

PRIORITY

The present application is a continuation of U.S. application Ser. No.10/120,687, filed Apr. 11, 2002 which is a continuation-in-partapplication of U.S. application Ser. No. 09/731,261, filed Dec. 6, 2000,and is also a continuation-in-part application of U.S. application Ser.No. 09/963,875, filed Sep. 26, 2001. The present application also claimspriority to U.S. Application Ser. No. 60/169,082, filed Dec. 6, 1999,U.S. Application Ser. No. 60/215,109, filed Jun. 28, 2000, and U.S.Application Ser. No. 60/238,880, filed Oct. 6, 2000.

The invention was made at least in part using U.S. government funds,grants DK30457, DK30834, DK55365, DK60125, awarded by the NationalInstitutes of Health, and therefore the U.S. government may retaincertain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

The invention is related to the field of stem cells and theirdifferentiation. In particular, it is related to the field of beta cellsof the islets of Langerhans in the pancreas and nestin positive liverstem cells and their differentiation from stem cells or progenitorcells, and the use of pancreatic stem cells, progenitor cells, anddifferentiated beta cells or nestin positive liver stem cells orprogenitor cells in transplantation.

BACKGROUND OF THE INVENTION

The origin of pancreatic islet cells, both during embryonic developmentand in a mature mammal, has remained uncertain despite intensive study.Certain ductal epithelial cells are capable of either differentiation ortransdifferentiation to form beta cells and other cell types found inmature islets (Bouwens, 1998). Ductal cells from isolated islets canproliferate in culture and, if transplanted into an animal, candifferentiate into functional beta cells (Cornelius et al., 1997).

It has been demonstrated that exendin-4, a long acting GLP-1 agonist,stimulates both the differentiation of β-cells from ductal progenitorcells (neogenesis) and proliferation of β-cells when administered torats. In a partial pancreatectomy rat model of type 2 diabetes, thedaily administration of exendin-4 for 10 days post pancreatectomyattenuated the development of diabetes. It has also been demonstratedthat exendin-4 stimulates the regeneration of the pancreas and expansionof β-cell mass by neogenesis and proliferation of β-cells (Xu et al.,1999, Diabetes, 48:2270-2276).

Ramiya et al. have demonstrated that islets generated in vitro frompluripotent stem cells isolated from the pancreatic ducts of adultprediabetic non-obese diabetic (NOD) mice differentiate to form glucoseresponsive islets that can reverse insulin-dependent diabetes afterbeing implanted, with or without encapsulation, into diabetic NOD mice(Ramiya et al., 2000, Nature Med., 6:278-282).

The insulinotropic hormone glucagon-like peptide (GLP)-1 which isproduced by the intestine, enhances the pancreatic expression of thehomeodomain transcription factor IDX-1 that is critical for pancreasdevelopment and the transcriptional regulation of the insulin gene.Concomitantly, GLP-1 administered to diabetic mice stimulates insulinsecretion and effectively lowers their blood sugar levels. GLP-1 alsoenhances β-cell neogenesis and islet size (Stoffers et al., 2000,Diabetes, 49:741-748).

Ferber et al. have demonstrated that adenovirus-mediated in vivotransfer of the PDX-1 (also known as IDX-1) transgene to mouse liverresults in the transconversion of a hepatocyte subpopulation towards aβ-cell phenotype. It has been demonstrated that after intravenousinfusion of mice with the PDX-1 adenoviral vector, up to 60% ofhepatocytes synthesized PDX-1. Although 60% of liver cells becameinfected with the adenovirus and expressed PDX-1, only a subset of 5-8%of these cells turned into insulin expressing beta cells. Theconcentration of immunoreactive insulin was increased in the liver andserum of treated mice. Mice treated with PDX-1 survivestreptozotocin-induced diabetes, and can even normalize glycemia (Ferberet al., 2000, Nature Med., 6:568-572).

While ductal cell cultures obtained from isolated islets apparentlycontain cells that can give rise to insulin-secreting cells, it hasremained unclear whether those cells represent true stem cells or merelyductal epithelial cells undergoing transdifferentiation. Even if suchpreparations contain genuine stem cells, it is unknown what fractionrepresent stem cells and what contaminating cell types may be present.There is a need in the art for the isolation of specific cell types frompancreatic tissue, the cell types being characterized as stem cellsusing molecular markers and demonstrated to be pluripotent and toproliferate long-term.

Pluripotent stem cells that are capable of differentiating into neuronaland glial tissues have been identified in brain. Neural stem cellsspecifically express nestin, an intermediate filament protein (Lendahlet al., 1990; Dahlstrand et al., 1992). Nestin is expressed in theneural tube of the developing rat embryo at day E11, reaches maximumlevels of expression in the cerebral cortex at day E16, and decreases inthe adult cortex, becoming restricted to a population of ependymal cells(Lendahl et al., 1990). Developing neural and pancreatic islet cellsexhibit phenotypic similarities characterized by common cellularmarkers.

The invention relates to a population of pancreatic isletstem/progenitor cells (IPCs) that are similar to neural and hepatic stemcells and differentiate into islet α-cells (glucagon) and β-cells(insulin). The invention also relates to nestin-positive liver cells.IPCs according to the invention are immunologicallysilent/immunoprivileged and are recognized by a transplant recipient asself. The IPCs according to the invention can be used for engraftmentacross allogeneic and xenogeneic barriers.

There is a need in the art for a method of engrafting stem cells acrossallogeneic and xenogeneic barriers.

There is also a need in the art for a method of treating type I diabetesmellitus wherein islets, nestin-positive pancreatic stem cells ornestin-positive liver stem cells are transferred into a recipient acrossallogeneic or xenogeneic barriers and graft rejection does not occur.

There is also a need in the art for a method of transplantation into amammal wherein islets, nestin-positive pancreatic stem cells ornestin-positive liver stem cells are transferred into a recipient acrossallogeneic or xenogeneic barriers and graft rejection does not occur.

SUMMARY OF THE INVENTION

It is an object of the invention to provide mammalian pancreatic orliver stem cells for use in treating diabetes mellitus and otherdisorders. It is also an object of the invention to provide methods foridentifying, localizing, and isolating pancreatic stem cells. It is afurther object of the invention to provide methods for differentiatingpancreatic stem cells to obtain cells that produce insulin and otherhormones. It is also an object of the invention to provide methods fortransplantation into a mammal that utilize mammalian pancreatic or liverstem cells. These and other objects of the invention are provided by oneor more of the embodiments described below.

One embodiment of the invention provides a method of treating a patientwith diabetes mellitus. A nestin-positive pancreatic stem cell isisolated from a pancreatic islet of a donor. In another embodiment, apancreatic stem cell that is positive for at least one of ABCG2, Oct3/4,GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunitsα6 and β1, C-kit, MDR-1, SUR-1, Kir 6.2 and/or does not express one orall of CD34, CD45, CD133, MHC class I and MHC class II is isolated froma pancreatic islet of a donor. In another embodiment, a pancreatic stemcell that is positive for SST-R2, SST-R3 and/or SST-R4 is isolated froma pancreatic islet of a donor. The stem cell is transferred into thepatient, where it differentiates into an insulin-producing cell.

Another embodiment provides another method of treating a patient withdiabetes. A nestin-positive pancreatic stem cell is isolated from apancreatic islet of a donor and expanded ex vivo to produce a progenitorcell. In another embodiment, a pancreatic stem cell that is positive forat least one of ABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2),Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2,SST-R3, SST-R4, SUR-1, Kir 6.2 and/or does not express one or all ofCD34, CD45, CD133, MHC class I and MHC class II is isolated from apancreatic islet of a donor and expanded ex vivo to produce a progenitorcell. The progenitor cell is transferred into the patient, where itdifferentiates into an insulin-producing beta cell. Another embodimentprovides still another method of treating a diabetes patient. Anestin-positive pancreatic stem cell is isolated from a pancreatic isletof a donor and expanded to produce a progenitor cell. In anotherembodiment, a pancreatic stem cell that is positive for at least one ofABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin,Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2, SST-R3, SST-R4-SUR-1, Kir 6.2 and/or does not express one or all of CD34, CD45, CD133,MHC class I and MHC class II is isolated from a pancreatic islet of adonor and expanded to produce a progenitor cell. The progenitor cell isdifferentiated in culture to form pseudo-islet like aggregates that aretransferred into the patient.

Another embodiment provides another method of treating a patient withdiabetes mellitus. A nestin-positive pancreatic stem cell is isolatedfrom a pancreatic islet of a donor and cultured ex vivo to produce aprogenitor cells. In another embodiment, a pancreatic stem cell that ispositive for at least one of ABCG2, Oct3/4, GLP-1 receptor, latrophilin(type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1,SST-R2, SST-R3, SST-R4, SUR-1, Kir 6.2 and/or does not express one orall of CD34, CD45, CD133, MHC class I and MHC class II is isolated froma pancreatic islet of a donor and cultured ex vivo to produce aprogenitor cell. The progenitor cell is transferred into the patient,where it differentiates into an insulin-producing beta cell.

In these embodiments, the patient can also serve as the donor of thepancreatic islet tissue, providing an isograft of cells ordifferentiated tissue.

In another preferred embodiment, prior to the step of transferring, thestem cell is treated ex vivo with an agent selected from the groupconsisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4,IDX-1, a nucleic acid molecule encoding IDX-1, betacellulin, activin A,TGF-β, and combinations thereof.

In another preferred embodiment, the step of transferring is performedvia endoscopic retrograde injection or injection into the pancreaticartery.

In another preferred embodiment, the method of treating a patient withdiabetes mellitus additionally comprises the step of treating thepatient with an immunosuppressive agent.

In another preferred embodiment, the immunosuppressive agent is selectedfrom the group consisting of FK-506, cyclosporin, and GAD65 antibodies.

Another embodiment provides a method of isolating a stem cell from apancreatic islet of Langerhans. A pancreatic islet is removed from adonor, and cells are cultured from it. A nestin-positive stem cell cloneis selected from the culture. In another embodiment, a clone ofpancreatic stem cells that is positive for at least one of ABCG2,Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrinsubunits α6 and β1, C-kit, MDR-1, SST-R2, SST-R3, SST-R4, SUR-1, Kir 6.2and/or do not express one or all of CD34, CD45, CD133, MHC class I andMHC class II is selected from the culture. Optionally, the islet isfirst purged of non-islet cells by culturing in a vessel coated withconcanavalin A, which binds the non-islet cells.

In a preferred embodiment, the method of isolating a stem cell furthercomprises the additional step of expanding the clone by treatment withan agent selected from the group consisting of EGF, bFGF-2, highglucose, KGF, HGF/SF, GLP-1, exendin-4, IDX-1, a nucleic acid moleculeencoding IDX-1, betacellulin, activin A, TGF-β, and combinationsthereof.

A further embodiment provides a method of inducing the differentiationof a nestin-positive pancreatic stem cell into a pancreatic progenitorcell. Another embodiment provides a method of inducing thedifferentiation of a pancreatic stem cell that is positive for at leastone of ABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1,Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2, SST-R3,SST-R4, SUR-1, Kir 6.2 and/or does not express one or all of CD34, CD45,CD133, MHC class I and MHC class II. As used herein, “differentiation”refers to the process by which a cell undergoes a change to a particularcell type, e.g. to a specialized cell type. The stem cell is treatedwith an agent selected from the group consisting of EGF, bFGF-2, highglucose, KGF, HGF/SF, IDX-1, a nucleic acid molecule encoding IDX-1,GLP-1, exendin-4, betacellulin, activin A, TGF-β, and combinationsthereof. The stem cell subsequently differentiates into a pancreaticprogenitor cell.

In a preferred embodiment, the pancreatic progenitor subsequently formspseudo-islet like aggregates.

Yet another embodiment provides an isolated, nestin-positive humanpancreatic or liver stem cell. Yet another embodiment provides anisolated pancreatic stem cell that is positive for at least one ofABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin,Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2, SST-R3, SST-R4,SUR-1, Kir 6.2 and/or does not express one or all of CD34, CD45, CD133,MHC class I and MHC class II. In versions of this embodiment, the stemcell differentiates into either a beta cell, an alpha cell, apseudo-islet like aggregate, or a hepatocyte. In versions of thisembodiment, the stem cell is immunoprivileged. In versions of thisembodiment, the stem cell does not express class I MHC antigens. Inversions of this embodiment, the stem cell does not express class II MHCantigens. In versions of this embodiment, the stem cell does not expressclass I or class II MHC antigens.

Still another embodiment provides a method of identifying a pancreaticcell as a stem cell. A cell is contacted with a labeled nestin-specificantibody. If the cell becomes labeled with the antibody, then the cellis identified as a stem cell. In another embodiment, a pancreatic stemcell is identified as a stem cell by contacting the cell with anantibody specific for any of ABCG2, Oct3/4, GLP-1 receptor, latrophilin(type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1,SST-R2, SST-R3, SST-R4, SUR-1 or Kir 6.2 wherein a cell that becomeslabeled with an antibody is identified as a stem cell. Optionaladditional steps include contacting the cell with an antibody tocytokeratin 19 and an antibody to collagen IV; the cell is identified asa stem cell if it does not become labeled with either the cytokeratin 19or the collagen IV antibody.

In another embodiment, a pancreatic stem cell is identified as a stemcell by contacting the cell with an antibody specific for any of CD34,CD35, CD133, MHC Class I and MHC Class II, wherein the cell isidentified as a stem cell if it does not become labeled with any of theantibodies.

Another embodiment provides a method of inducing a nestin-positivepancreatic stem cell to differentiate into hepatocytes. Thenestin-positive pancreatic stem cell is treated with an effective amountof an agent that induces the stem cell to differentiate into hepatocytesor into progenitor cells that differentiate into hepatocytes. In apreferred embodiment, the agent is cyclopamine. Another embodimentprovides a method of inducing a pancreatic stem cell that is positivefor at least one of ABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2),Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2,SST-R3, SST-R4, SUR-1, Kir 6.2 and/or does not express one or all ofCD34, CD45, CD133, MHC class I and MHC class II to differentiate intohepatocytes by treatment with an effective amount of an agent thatinduces the stem cell to differentiate into hepatocytes or intoprogenitor cells that differentiate into hepatocytes.

Yet another embodiment provides a method of treating a patient withliver disease. A nestin-positive pancreatic stem cell is isolated from apancreatic islet of a donor and transferred into the patient, where thestem cell differentiates into a hepatocyte. In another embodiment, apancreatic stem cell that is positive for at least one of ABCG2, Oct3/4,GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunitsα6 and β1, C-kit, MDR-1, SST-R2, SST-R3, SST-R4, SUR-1, Kir 6.2 and/ordoes not express one or all of CD34, CD45, CD133, MHC class I and MHCclass II is isolated from a pancreatic islet of a donor and transferredinto the patient where the stem cell differentiates into a hepatocyte.

In a related embodiment, the stem cell is expanded ex vivo to aprogenitor cell, which is transferred into the patient and furtherdifferentiates into a hepatocyte.

In another related embodiment, the stem cell is differentiated ex vivoto a progenitor cell, which is transferred into the patient and furtherdifferentiates into a hepatocyte. In another related embodiment, thestem cell is differentiated ex vivo into hepatocytes, which aretransplanted into the patient.

In these embodiments, the patient can also serve as the donor of thepancreatic islet tissue, providing an isograft of cells ordifferentiated tissue.

Yet another embodiment provides an isolated, nestin-positive human liverstem cell. In versions of this embodiment, the stem cell isimmunoprivileged. In versions of this embodiment, the stem cell does notexpress class I MHC antigens. In versions of this embodiment, the stemcell does not express class II MHC antigens. In versions of thisembodiment, the stem cell does not express class I or class II MHCantigens.

Yet another embodiment provides an isolated, nestin-positive human stemcell that is not a neural stem cell, that is capable of transplant intoan animal without causing graft versus host rejection. In versions ofthis embodiment, the stem cell is not major histocompatibility complexclass I or class I restricted. Still another embodiment provides anisolated human stem cell that is positive for at least one of ABCG2,Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrinsubunits α6 and β1, C-kit, MDR-1, SST-R2, SST-R3, SST-R4, SUR-1, Kir 6.2and/or does not express one or all of CD34, CD45, CD133, MHC class I andMHC class II, that is not a neural stem cell, that is capable oftransplant into an animal without causing graft versus host rejection.

A “stem cell” as used herein is a undifferentiated cell which is capableof essentially unlimited propagation either in vivo or ex vivo andcapable of differentiation to other cell types. This can be to certaindifferentiated, committed, immature, progenitor, or mature cell typespresent in the tissue from which it was isolated, or dramaticallydifferentiated cell types, such as for example the erythrocytes andlymphocytes that derive from a common precursor cell, or even to celltypes at any stage in a tissue completely different from the tissue fromwhich the stem cell is obtained. For example, blood stem cells maybecome brain cells or liver cells, neural stem cells can become bloodcells, such that stem cells are pluripotential, and given theappropriate signals from their environment, they can differentiate intoany tissue in the body.

In one embodiment, a “stem cell” according to the invention isimmunologically blinded or immunoprivileged. As used herein,“immunologically blinded” or “immunoprivileged” refers to a cell thatdoes not elicit an immune response. As used herein, an “immune response”refers- to a response made by-the immune system to a foreign substance.An immune response, as used herein, includes but is not limited totransplant or graft rejection, antibody production, inflammation, andthe response of antigen specific lymphocytes to antigen. An immuneresponse is detected, for example, by determining if transplantedmaterial has been successfully engrafted or rejected, according tomethods well-known in the art, and as defined herein in the sectionentitled, “Analysis of Graft Rejection”. In one embodiment, an“immunogically blinded stem cell” or an “immunoprivileged stem cell”according to the invention can be allografted or xenografted withouttransplant rejection, and is recognized as self in the transplantrecipient or host.

Transplanted or grafted material can be rejected by the immune system ofthe transplant recipient or host unless the host is immunotolerant tothe transplanted material or unless immunosupressive drugs are used toprevent rejection.

As used herein, a host that is “immunotolerant”, according to theinvention, fails to mount an immune response, as defined herein. In oneembodiment, a host that is “immunotolerant” does not reject or destroytransplanted material. In one embodiment, a host that is“immunotolerant” does not respond to an antigen by producing antibodiescapable of binding to the antigen.

As used herein, “rejection” refers to rejection of transplanted materialby the immune system of the host. In one embodiment, “rejection means anoccurrence of more than 90% cell or tissue necrosis of the transplantedmaterial in response to the immune response of the host. In anotherembodiment, “rejection” means a decrease in the viability such that theviability of the transplanted material is decreased by 90% or more ascompared to the viability of the transplanted material prior totransplantation, in response to the immune response of the host. Adecrease in viability can be determined by methods well known in theart, including but not limited to trypan blue exclusion staining. Inanother embodiment, “rejection” means failure of the transplantedmaterial to proliferate. Proliferation can be measured by methods knownin the art including but not limited to hematoxylin/eosin staining. Theoccurrence of transplant rejection and/or the speed at which rejectionoccurs following transplantation will vary depending on factors,including but not limited to the transplanted material (i.e., the celltype, or the cell number) or the host (i.e., whether or not the host isimmunotolerant and/or has been treated with an immunosuppressive agent.As used herein, “graft versus host rejection” or “graft versus hostresponse” refers to a cell-mediated reaction in which T-cells of thetransplanted material react with antigens of the host.

As used herein, “host versus graft rejection” or “host versus graftresponse” refers to a cell-mediated reaction in which cells of thehost's immune system attack the foreign grafted or transplantedmaterial.

In another embodiment of the invention, an immune response has occurredif production of a specific antibody (for example an antibody that bindsspecifically to an antigen on the transplanted material, or an antibodythat binds specifically to the foreign substance or a product of theforeign substance) is detected by immunological methods well-known inthe art, including but not limited to ELISA, immunostaining,immunoprecipitation and Western Blot analysis.

Stem cells express morphogenic or growth hormone receptors on the cellsurface, and can sense, for example, injury-related factors thenlocalize to and take residence at sites of tissue injury, or sense theirlocal microenvironment and differentiate into the appropriate cell type.

“Essentially unlimited propagation” can be determined, for example, bythe ability of an isolated stem cell to be propagated through at least50, preferably 100, and even up to 200 or more cell divisions in a cellculture system. Stem cells can be “totipotent,” meaning that they cangive rise to all the cells of an organism as for germ cells.“Totipotent” also means the fertilized egg that can give rise to bothembryo and trophoblast. Stem cells can also be “pluripotent,” meaningthat they can give rise to many different cell types, but not all thecells of an organism. “Pluripotent” also means gives rise to embryo onlyand not to trophoblast. When a stem cell differentiates it generallygives rise to a more adult cell type, which may be a partiallydifferentiated cell such as a progenitor cell, a differentiated cell-,-or a terminally differentiated cell. Stem cells can be highly motile.

“Nestin” refers to an intermediate filament protein having a sequencedisclosed in Genbank Access No. X65964 (FIG. 7).

“ABCG2” refers to ATP-binding cassette multidrug resistance transporterG2 having a sequence disclosed in Genbank Access No. XM_(—)032424 (FIG.18). One of skill in the art will recognize that equivalents existwherein the DNA sequence encoding ABCG2 may vary, but wherein theencoded amino acid sequence remains the same.

“Oct3/4” refers to a POU/Homeodomain transcription factor having asequence disclosed in Genbank Access No. NM 013633.

“GLP-1R” refers to the glucagon-like peptide-1-receptor encoded by thenucleic acid sequence shown in FIG. 21 (GenBank Accession No. U01156),and having the amino acid sequence also shown in FIG. 17 (GenBankAccession No. U01156). One of skill in the art will recognize thatequivalents exist wherein the DNA sequence encoding GLP-1R may vary, butwherein the encoded amino acid sequence remains the same.

“Latrophilin (type 2)” refers to a G protein-coupled receptor having asequence disclosed in Genbank Access No. AJ131581.

“Hes-1” refers to a bHLH transcription factor having a sequencedisclosed in Genbank Access No. NM_(—)005524.

Integrins are integral cell-surface proteins composed of an alpha chainand a beta chain. A given chain may combine with multiple partnersresulting in different integrins. For example, alpha 6 may combine withbeta 4 in the integrin referred to as TSP180, or with beta 1 in theintegrin VLA-6.

The integrin subunit “α6” refers to the human integrin α6 cDNA having asequence disclosed in Genbank Access No. NM_(—)000210.

The integrin subunit “β1” refers to the human β1 cDNA having a sequencedisclosed in Genbank Access No.: X07979.

“c-Kit” refers to a cell surface receptor tyrosine kinase having asequence disclosed in Genbank Access No. X06182.

“MDR-1” refers to the multidrug resistance transporter 1 having asequence disclosed in Genbank Access No. NM_(—)000927.

“SST-R2” SST-R3” and “SST-R4” refer to somatostatin receptors having asequence disclosed in Genbank Access Nos. XM_(—)085745 (SSTR-2);NM_(—)001051 (SSTR-3); XM_(—)009594 (SSTR-4).

“SUR-1” refers to a human sulfonylurea receptor having a sequencedisclosed in Genbank Access No. XM_(—)042740.

“Kir 6.2” refers to a human inward rectifier potassium channel having asequence disclosed in Genbank Access No. AF021139.

“CD34” refers to a transmembrane glycoprotein having a sequencedisclosed in Genbank Access No. NM_(—)001773.

“CD45” refers to the leukocyte common antigen having a sequencedisclosed in Genbank Access No. Y00638.

“CD133” refers to a five-transmembrane hematopoietic stem cell antigenhaving a sequence disclosed in Genbank Access No. NM_(—)006017.

A “pancreatic” stem cell means a stem cell that has been isolated frompancreatic tissue and/or a cell that has all of the characteristics of:nestin-positive staining, nestin gene expression, cytokeratin-19negative staining, long-term proliferation in culture, and the abilityto differentiate into pseudo-islets in culture.

A “pancreatic” stem cell also refers to a stem cell that is positive forat least one of ABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2),Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, Kir 6.2and/or does not express one or all of CD34, CD45, CD133, MHC class I andMHC class II.

In one embodiment, a “pancreatic” stem cell also refers to a stem cellthat is positive for SST-R2, SST-R3 and/or SST-R4.

A “liver” stem cell means a stem cell that has been isolated from livertissue and/or a cell that has all of the characteristics of:nestin-positive staining, nestin gene expression, and long-termproliferation in culture.

A “progenitor cell” is a cell that is derived from a stem cell bydifferentiation and is capable of further differentiation to more maturecell types.

As used herein, the term “insulin-producing beta cell” refers to anycell which can produce and secrete insulin in a similar amount to thatproduced and secreted by a beta cell of the islets of Langerhans in thehuman pancreas. Preferably, the secretion of insulin by aninsulin-producing beta cell is also regulated in a similar fashion tothe regulation of insulin secretion by a human beta cell in situ; forexample, insulin secretion should be stimulated by an increase in theglucose concentration in the solution surrounding the insulin-producingbeta cell.

“Pseudo-islet like” aggregates are artificial aggregates ofinsulin-secreting cells which resemble in form and function the isletsof Langerhans of the pancreas. Pseudo-islet like aggregates are createdex vivo under cell culture conditions. They are approximately 50-150 μmin diameter (compared to an average diameter of 100 μm for in situislets) and spheroid in form.

“Isolating” a stem cell refers to the process of removing a stem cellfrom a tissue sample and separating away other cells which are not stemcells of the tissue. An isolated stem cell will be generally free fromcontamination by other cell types and will generally have the capabilityof propagation and differentiation to produce mature cells of the tissuefrom which it was isolated. However, when dealing with a collection ofstem cells, e.g., a culture of stem cells, it is understood that it ispractically impossible to obtain a collection of stem cells which is100% pure. Therefore, an isolated stem cell can exist in the presence ofa small fraction of other cell types which do not interfere with theutilization of the stem cell for analysis or production of other,differentiated cell types. Isolated stem cells will generally be atleast 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% pure.Preferably, isolated stem cells according to the invention will be atleast 98% or at least 99% pure.

A stem cell is “expanded” when it is propagated in culture and givesrise by cell division to other stem cells and/or progenitor cells.Expansion of stem cells may occur spontaneously as stem cellsproliferate in a culture or it may require certain growth conditions,such as a minimum cell density, cell confluence on the culture vesselsurface, or the addition of chemical factors such as growth factors,differentiation factors, or signaling factors.

A stem cell, progenitor cell, or differentiated cell is “transplanted”or “introduced” into a mammal when it is transferred from a culturevessel into a patient. Transplantation, as used herein, can include thesteps of isolating a stem cell according to the invention andtransferring the stem cell into a mammal or a patient. Transplantationcan involve transferring a stem cell into a mammal or a patient byinjection of a cell suspension into the mammal or patient, surgicalimplantation of a cell mass into a tissue or organ of the mammal orpatient, or perfusion of a tissue or organ with a cell suspension. Theroute of transferring the stem cell or transplantation, will bedetermined by the need for the cell to reside in a particular tissue ororgan and by the ability of the cell to find and be retained by thedesired target tissue or organ. In the case where a transplanted cell isto reside in a particular location, it can be surgically placed into atissue or organ or simply injected into the bloodstream if the cell hasthe capability to migrate to the desired target organ.

Transplantation, as used herein, can include the steps of isolating astem cell according to the invention, and culturing and transferring thestem cell into a mammal or a patient. Transplantation, as used herein,can include the steps of isolating a stem cell according to theinvention, differentiating the stem cell, and transferring the stem cellinto a mammal or a patient. Transplantation, as used herein, can includethe steps of isolating a stem cell according to the invention,differentiating and expanding the stem cell and transferring the stemcell into a mammal or a patient.

Treatment with an immunosuppressive agent can be accomplished byadministering to a patient in need thereof any agent which prevents,delays the occurrence of or decreases the intensity of the desiredimmune response, e.g., rejection of a transplanted cell, tissue, ororgan.

As used herein, “immunosuppression” refers to prevention of the immuneresponse (for example by the administration of an “immunosuppresiveagent”, as defined herein) such that an “immune response”, as definedherein, is not detectable. As used herein, “prevention” of an immuneresponse means an immune response is not detectable. An immune response(for example, transplant rejection or antibody production) is detectedaccording to methods well-known in the art and defined herein.

“Immunosuppression” according to the invention also means a delay in theoccurrence of the immune response as compared to any one of a transplantrecipient that has not received an immunosuppresive agent, or atransplant recipient that has been transplanted with material that isnot “immunologically blinded” or “immunoprivileged”, as defined herein.A delay in the occurrence of an immune response can be a short delay,for example 1 hr-10 days, i.e., 1 hr, 2, 5 or 10 days. A delay in theoccurrence of an immune response can also be a long delay, for example,10 days-10 years (i.e., 30 days, 60 days, 90 days, 180 days, 1, 2, 5 or10 years).

“Immunosuppression” according to the invention also means a decrease inthe intensity of an immune response. According to the invention, theintensity of an immune response can be decreased such that it is 5-100%,preferably, 25-100% and most preferably 75-100% less than the intensityof the immune response of any one of a transplant recipient that has notreceived an immunosuppresive agent, or a transplant recipient that hasbeen transplanted with material that is not “immunologically blinded” or“immunoprivileged”, as defined herein. The intensity of an immuneresponse can be measured by determining the time point at whichtransplanted material is rejected. For example, an immune responsecomprising rejection of transplanted material at day 1,post-transplantation, is of a greater intensity than an immune responsecomprising the rejection of transplanted material at day 30,post-transplantation. The intensity of an immune response can also bemeasured by quantitating the amount of a particular antibody capable ofbinding to the transplanted material, wherein the level of antibodyproduction correlates directly with the intensity of the immuneresponse. Alternatively, the intensity of an immune response can bemeasured by determining the time point at which a particular antibodycapable of binding to the transplanted material is detected.

Various strategies and agents can be utilized for immunosuppression. Forexample, the proliferation and activity of lymphocytes can be inhibitedgenerally with agents such as, for example, FK-506, or cyclosporin orother immunosuppressive agents. Another possible strategy is toadminister an antibody, such as an anti-GAD65 monoclonal antibody, oranother compound which masks a surface antigen on a transplanted celland therefore renders the cell practically invisible to the immunesystem of the host.

An “immunosuppressive agent” is any agent that prevents, delays theoccurrence of or reduces the intensity of an immune reaction against aforeign cell in a host, particularly a transplanted cell. Preferred areimmunosuppressive agents which suppress cell-mediated immune responsesagainst cells identified by the immune system as non-self. Examples ofimmunosuppressive agents include but are not limited to cyclosporin,cyclophosphamide, prednisone, dexamethasone, methotrexate, azathioprine,mycophenolate, thalidomide, FK-506, systemic steroids, as well as abroad range of antibodies, receptor agonists, receptor antagonists, andother such agents as known to one skilled in the art.

A “mitogen” is any agent that stimulates mitosis and cell proliferationof a cell to which the agent is applied.

A “differentiation factor” is any agent that causes a stem cell orprogenitor cell to differentiate into another cell type. Differentiationis usually accomplished by altering the expression of one or more genesof the stem cell or progenitor cell and results in the cell altering itsstructure and function.

A “signaling factor” as used herein is an agent secreted by a cell whichhas an effect on the same or a different cells. For example, a signalingfactor can inhibit or induce the growth, proliferation, ordifferentiation of itself, neighboring cells, or cells at distantlocations in the organism. Signaling factors can, for example, transmitpositional information in a tissue, mediate pattern formation, or affectthe size, shape and function of various anatomical structures.

As used herein, a mammal refers to any mammal including but not limitedto human, mouse, rat, sheep, monkey, goat, rabbit, hamster, horse, cowor pig.

A “non-human mammal”, as used herein, refers to any mammal that is not ahuman.

As used herein, “allogeneic” refers to genetically different members ofthe same species.

As used herein, “isogeneic” refers to of an identical geneticconstitution.

As used herein, “xenogeneic” refers to members of a different species.

As used herein, “culturing” refers to propagating or nurturing a cell,collection of cells, tissue, or organ, by incubating for a period oftime in an environment and under conditions which support cell viabilityor propagation. Culturing can include one or more of the steps ofexpanding and proliferating a cell, collection of cells, tissue, ororgan according to the invention.

The invention also provides for a pharmaceutical composition comprisingthe isolated stem cells of the invention admixed with a physiologicallycompatible carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show dual fluorescence immunocytochemical staining ofrat pancreatic islets at embryonic day 16 (FIG. 1A) and at day 60 afterbirth (FIG. 1B). Immunostaining with an antibody for nestin is shown inwhite (red in the original, with Cy3 as fluorophore) and with anantibody for insulin is shown in grey (green in the original, with Cy2as fluorophore).

FIG. 2 shows the result of RT-PCR performed using mRNA obtained from 50rat islets. Forward and reverse primers are indicated. The single bandof 834 bp was sequenced and identified substantially as the sequence fornestin.

FIG. 3 shows nestin-positive cells that have proliferated out from acultured rat islet.

FIG. 4 shows the development of islet like structures in culture.

FIG. 5 shows the results of RT-PCR analysis of islet-like structuresgenerated in culture. Expression of NCAM and cytokeratin-19 (CK19) wasdetected.

FIG. 6 shows the stimulation of nestin mRNA expression by high glucose.APRT was examined as a control.

FIG. 7 is the amino acid and nucleotide sequences of nestin.

FIG. 8 depicts expression of the neural stem cell-specific marker nestinin a distinct cell population within pancreatic islets as determined byimmunocytochemistry or RT-PCR.

FIG. 9 depicts characterization of nestin in stem cells isolated fromthe pancreas by immunocytochemistry and RT-PCR.

FIG. 10 depicts expression of homeodomain protein IDX-1 and proglucagonin human islet-like clusters derived from nestin-positive isletprogenitor cells (NIPs).

FIG. 11 demonstrates localization of nestin-positive cells to localizedregions of the ducts of the rat pancreas.

FIG. 12 depicts alternative models for the origin of pancreatic ductcells that are progenitors of islet endocrine cells.

FIG. 13A and B depicts immunofluorescent staining of nestin positiveliver stem cells.

FIG. 14 depicts the sequential appearance of transcription factorsduring development of the murine endocrine pancreas.

FIG. 15 depicts expression of neuroendocrine, exocrine pancreatic andhepatic markers in human NIP cultures containing stem cells.

FIG. 16 depicts expression of proglucagon and insulin mRNA as determinedby RT-PCR and insulin secretion.

FIG. 17 depicts NIP markers.

FIG. 18 depicts the nucleic acid(a) and amino acid(b) sequence of humanABCG2.

FIG. 19 a depicts expression of the ATP-binding cassette transporterABCG2 by RT-PCR and Southern blot hybridization.

FIG. 19 b demonstrate that nestin-positive islet derived progenitorcells (NIPs) include a significant number of SP cells as demonstrated byHoechst 33342 staining.

FIG. 19 c demonstrates that dye efflux from SP cells is inhibited in thepresence of verapamil. SP-gated cells are 2.1% of the total number ofanalyzed cells in B versus 0.1% in C.

FIG. 20 demonstrates that SP cells isolated by FACS co-express highlevels of ABCG2, MDR1 and nestin. FIG. 20A depicts that SP cells andnon-SP control cells were isolated by FACS after Hoechst 33342 staining(R1 and R2, respectively). FIG. 20B demonstrates that the cells wereanalyzed for the expression of ABCG2, MDR1, nestin, and GAPDH RNA byRT-PCR. The identity of the PCR products for ABCG2 and MDR1 wasconfirmed by Southern blot hybridization.

FIG. 21 shows the nucleic acid (21A) and amino acid (21B) sequence ofthe human GLP-1R.

FIG. 22 shows the expression of GLP-1R on pancreatic islet-derivedStem/progenitor cells. Panel A shows the immunocytochemical detection ofGLP-1R (Cy-3) and nuclei stained with DAPI. Panel B shows RT-PCR of RNAprepared from NIPS with primers specific for GLP-1R.

FIG. 23 shows GLP-1 (7-36) amide and tolbutanide stimulation of Ca²⁺influx in nestin-positive NIPs.

FIG. 24 shows immunohistochemical staining of NIPs demonstrating GLP-1induced differentiation. Panel A shows immunohistochemicalidentification of nestin and insulin. Panel B shows nestin and insulinimmunohistochemical staining after cells were challenged with GLP-1.

FIG. 25 shows the levels of insulin secretion from NMP cultureschallenged with GLP-1.

FIG. 26 shows the immunohistochemical analysis of insulin expression inNIP cultures transfected with human Idx-1.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have identified and isolated a special subclass ofductal cells from the islets of Langerhans of mammalian pancreas thathave the functional and molecular characteristics of stem cells. Inparticular, these newly discovered pancreatic stem cells arecharacterized inter alia by one or more (and preferably all of):nestin-positive staining, nestin gene expression, GLP-1R-positivestaining, GLP-1R gene expression, ABCG2 positive staining, ABCG2 geneexpression, Oct3/4 positive staining, Oct3/4 gene expression,latrophilin (type 2) positive staining, latrophilin (type 2) geneexpression, Hes-1 positive staining, Hes-1 gene expression, Integrinsubunits α6 and β1 positive staining, Integrin subunits α6 and β1 geneexpression, C-kit positive staining, C-kit gene expression, MDR-1positive staining, MDR-1 gene expression, SST-R, 2, 3, 4 positivestaining, SST-R, 2, 3, 4 gene expression, SUR-1 positive staining, SUR-1gene expression, Kir 6.2 positive staining, Kir 6.2 gene expression CD34negative staining, CD45 negative staining, CD133 negative staining, MHCclass I negative staining, MHC class II negative staining,cytokeratin-19 negative staining, long-term proliferation in culture,and the ability to differentiate into pseudo-islets in culture. Thepresent inventors have also identified liver cells that exhibitnestin-positive staining.

In one embodiment, the invention provides stem cells for a variety ofapplications, including but not limited to cellular replacement therapyfor type I insulin-dependent diabetes and other forms of diabetes aswell as the development of research tools to study the onset andprogression of various diabetic conditions, hormonal abnormalities, andgenetic diseases or conditions, such as the association of polymorphismswith particular physiologic or pathologic states. The stem cells of theinvention can also be used to carry out gene therapy of endocrinepancreatic or other tissues in isograft, allograft or xenografttransplantations. Further, the stem cells described herein can be usedto produce recombinant cells, artificial tissues, and replacement organsin culture. They can also be used for the ex vivo production of insulinand other hormones. Molecular characteristics of pancreatic stem cellsdiscovered by the inventors, such as nestin-positive, GLP-1R-positive,ABCG2 positive staining, Oct3/4 positive, latrophilin (type 2) positive,Hes-1 positive, Integrin subunits α6 and β1 positive, C-kit positive,MDR-1 positive, SST-R, 2, 3, 4 positive, SUR-1 positive, Kir 6.2positive and cytokeratin-19 negative, CD34 negative, CD45 negative,CD133 negative, MHC class I negative and MHC class II negative staining,or liver stem cells, such as nestin-positive staining, can be used invarious diagnostic, pathological, or investigative procedures toidentify, localize, and quantitate stem cells in tissues from a patientor experimental animal.

Identification of Stem Cells in Pancreatic Islets

Previous investigators have focused on ductal epithelial cells ofpancreatic islets or exocrine tissue as a possible source of stem cellsfor the neogenesis of islet endocrine cells. Nestin is an intermediatefilament protein that was cloned by screening a cDNA library from E15rat embryos with a monoclonal antibody named R.401 (Hockfield & McKay,1985; Lendahl et al., 1990). Nestin was primarily found inneuroepithelial stem cells and is expressed in the developing centralnervous system. After maximum levels are reached in-the rat embryo atE16, nestin expression declines to almost undetectable levels in adultcerebral cortex, coinciding with terminal differentiation of earlynestin-expressing progenitor cells (Lendahl et al., 1990). Nestin wasinitially found exclusively in stem cells of the embryonic developingbrain and skeletal muscle (Lendahl et al., 1990). Later studiesidentified nestin-positive neural stem cells in the subependymal layerof the adult mammalian forebrain (Morshead et al., 1994).Nestin-positive stem cells have been shown to be pluripotential evenwhen isolated from adult mice or rat brain. For example, nestin-positivestem cells can generate all three major classes of neural cells inculture: neurons, astrocytes, and oligodendrocytes (Reynolds & Weiss,1996). Nestin-positive neural stem cells respond to spinal cord injuryby proliferation and degeneration of migratory cells that differentiateinto astrocytes, participate in scar formation (Johansson et al., 1999)and restore hematopoietic cells of the bone marrow after infusion intoirradiated mice (Bjornson et al., 1999).

Characterization of Stem Cells

Stem cells according to the invention can be identified by theirexpression of nestin and/or GLP-1R, ABCG2, Oct3/4, latrophilin (type 2),Hes-1, Integrin subunits α6 and β1, C-kit, MDR-1, SST-R, 2, 3, 4, SUR-1and Kir 6.2, by, for example, FACS, immunocytochemical staining, RT-PCR,Southern Northern and Western blot analysis, and other such techniquesof cellular identification as known to one skilled in the art.

Immunocytochemical staining, for example, is carried out according tothe following method. Cryosections (6 μM) prepared from pancreata orliver, as well as cells, are fixed with 4% paraformaldehyde inphosphate. Cells are first blocked with 3% normal donkey serum for 30min at room temperature and incubated with a primary antisera to theprotein of interest overnight at 4° C. The antisera is rinsed off withPBS and incubated with the appropriate fluorescently labeled secondaryantisera for 1 hour at room temperature. Slides are then washed with PBSand coverslipped with fluorescent mounting medium (Kirkegaard and PerryLabs, Gaithersburg, Md.). Fluorescence images are obtained using a ZeissEpifluorescence microscope equipped with an Optronics TEC-470 CCD camera(Optronics Engineering, Goleta, Calif.) interfaced with a PowerMac 7100installed with IP Lab Spectrum analysis software (Signal Analytics Corp,Vienna, Va.).

Antisera useful according to the invention include the following: mousemonoclonal antibodies to human cytokeratin 19 (clone K4.62, Sigma, St.Louis, Mo.), rabbit polyclonal antisera to rat nestin and to IDX-1(prepared by immunizations of rabbits with a purified GST-nestin fusionprotein or the last twelve amino acids of rat IDX-1, respectively)(McManus et al., 1999, J. Neurosci., 19:9004-9015), rabbit polyclonalantisera to GLP-1R (Heller et al., 1997, Diabetes 46: 7851) antisera toABCG2 (MAB4146, Chemicon (Temecula, Calif.)) antisera to integrin αG(SC-6597, Santa Cruz), antisera to integrin β1, (SC-6622, Santa Cruz),antisera to HES 1 (AB5702, Chemicon), antisera to CD 45 (31252X, BDPharmingen (San Diego, Calif.)), antisera to CD 34 (MS-363-PO,NeoMarkers (Freemont, Calif.)), antisera to MHC I (MS-557-PO,NeoMarkers), antisera to MHC II (MS-162-PO, NeoMarkers) antisera toMDR-1 (p170) (MS-660-PO, NeoMarkers), antisera to Oct 3/4 (SC-5279,Santa Cruz (SC, CA)), antisera to SUR-1(SC-5789, Santa Cruz) antisera toKIR 6.2 (SC-11227, Santa Cruz), antisera to ABC G2 (SC-18841, SantaCruz) antisera to c-kit (SC-1493, Santa Cruz), antisera to SSTR 2(SC-11606, Santa Cruz), antisera to SSTR 3 (SC-11610, Santa Cruz)antisera to SSTR 4 (SC-11619, Santa Cruz), guinea pig anti-insulin andanti-pancreatic polypeptide antisera, obtained from Linco, St. Charles,Mo., and mouse antiglucagon and rabbit antisomatostatin antisera,purchased from Sigma (St. Louis, Mo.) and DAKO (Carpinteria, Calif.),respectively, mouse anti-human galanin (Peninsula Laboratories, Belmont,Calif.), collagen IV antisera (Caltag Laboratories, San Francisco,Calif.), mouse anti-rat MHC class I serum (Seroteck), and antirat MHCclass II serum. The invention contemplates that other antisera directedto such markers is available, or will be developed. Such other antiserais considered to be within the scope of the invention.

RT-PCR and Southern blot analysis are performed according to thefollowing methods. Total cellular RNA prepared from rat or human isletsis reverse transcribed and amplified by PCR for about 35 cyclesdepending on the desired degree of amplification, as describedpreviously (Daniel, et al., 1998, Endocrinology, 139:3721-3729).Oligonucleotides used as primers or amplimers for the PCR and as probesfor subsequent Southern blot hybridization are: Rat nestin: Forward,5′gcggggcggtgcgtgactac3′; Reverse, 5′aggcaagggggaagagaaggatgt3′;Hybridization, 5′aagctgaagccgaatttccttgggataccagagga3′. Rat keratin 19:Forward, 5′acagccagtacttcaagacc3′; Reverse, 5′ctgtgtcagcacgcacgtta3′;Hybridization, 5′tggattccacaccaggcattgaccatgcca3′. Rat NCAM: Forward,5′cagcgttggagagtccaaat3′; Reverse, 5′ttaaactcctgtggggttgg3′;Hybridization, 5′aaaccagcagcggatctcagtggtgtggaacgatgat3′. Rat IDX-1Forward, 5′atcactggagcagggaagt3′ Reverse, 5′gctactacgtttcttatct3′Hybridization, 5′gcgtggaaaagccagtggg3′ Human nestin: Forward,5′agaggggaattcctggag3′; Reverse, 5′ctgaggaccaggactctcta3′;Hybridization, 5′tatgaacgggctggagcagtctgaggaaagt3′. Human keratin:Forward, 5′cttttcgcgcgcccagcatt3′; Reverse, 5′gatcttcctgtccctcgagc3′;Hybridization, 5′aaccatgaggaggaaatcagtacgctgagg3′. Human glucagon:Forward, 5′atctggactccaggcgtgcc3′; Reverse, 5′agcaatgaattccttggcag3′;Hybridization, 5′cacgatgaatttgagagacatgctgaaggg3′; Human E-CadherinForward, 5′ agaacagcacgtacacagcc 3′ Reverse, 5′cctccgaagaaacagcaaga 3′Hybridization, 5′ tctcccttcacagcagaactaacacacggg 3′ Human transthyretinForward, 5′ gcagtcctgccatcaatgtg 3′ Reverse, 5′ gttggctgtgaataccacct 3′Hybridization, 5′ ctggagagctgcatgggctcacaactgagg 3′ Human PancreaticAmylase Forward, 5′gactttccagcagtcccata 3′ Reverse, 5′gtttacttcctgcagggaac 3′ Hybridization, 5′ttgcactggagaaggattacgtggcgttcta 3′ Human procarboxypeptidase Forward, 5′tgaaggcgagaaggtgttcc 3′ Reverse, 5′ ttcgagatacaggcagatat 3′Hybridization, 5′ agttagacttttatgtcctgcctgtgctca 3′ Human SynaptophysinForward, 5′ cttcaggctgcaccaagtgt 3′ Reverse, 5′ gttgaccatagtcaggctgg 3′Hybridization, 5′ gtcagatgtgaagatggccacagacccaga 3′ Human HepatocyteGrowth Factor (HGF) Forward, 5′ gcatcaaatgtcagccctgg 3′ Reverse, 5′caacgctgacatggaattcc 3′ Hybridization, 5′ tcgaggtctcatggatcatacagaatcagg3′ Human cMET (HGF-receptor) Forward, 5′ caatgtgagatgtctccagc 3′Reverse, 5′ ccttgtagattgcaggcaga 3′ Hybridization, 5′ggactcccatccagtgtctccagaagtgat 3′ Human XBP-1 Forward,5′gagtagcagctcagactgcc 3′ Reverse, 5′ gtagacctctgggagctcct 3′Hybridization, 5′ cgcagcactcagactacgtgcacctctgca 3′ Human Glut-2Forward, 5′ gcagctgctcaactaatcac 3′ Reverse, 5′ tcagcagcacaagtcccact 3′Hybridization, 5′ acgggcattcttattagtcagattattggt 3′ Human InsulinForward, 5′ aggcttcttctacaca3′ Reverse, 5′ caggctgcctgcacca 3′Hybridization, 5′ aggcagaggacctgca 3′

Other such sequences are possible and such sequences are considered tobe within the scope of the art. The invention includes oligonucleotidesused as primers or amplimers for the PCR and as probes for Southernanalysis for any of the markers selected from the group consisting ofABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin,Integrin subunits α6 and β1, C-kit, MDR-1, SST-R, 2, 3, 4, SUR-1, Kir6.2, CD34, CD45, CD133, MHC class I and MHC class II. As a generalguide, primers are selected from two different exons and encompass atleast one intronic sequence. In addition, an RT minus control is run formost samples. PCR amplification is effectuated at 94° C. for 1 minfollowed by 94° C. for 10 secs, 58/56° C. for 10 secs, 72° C. for 1 min,35 cycles, and 72° C. for 2 min. The annealing temperature is 58° C. forrat nestin and 56° C. for the remaining primer pairs.

For RT-PCR of mRNA isolated from a mammal that is not rat or human,oligonucleotides that are specific for the amplified nucleic acid fromthe mammalian species being analyzed are prepared. The selection and useof such primers is known to one skilled in the art.

For Southern hybridization oligonucleotide probes are labeled with anappropriate radionuclide, such as γ³²P ATP, using conventionaltechniques. Radiolabeled probes are hybridized to PCR productstransferred to nylon membranes at 37° C. for one hour, then washed in1×SSC+0.5% SDS at 55° C. for 10-20 min or 0.5×SCC+0.5% SDS at 42° forthe human PCR products.

Nestin as a Marker of Pancreatic Stem Cells

The inventors have now unexpectedly discovered that the pancreas ofadult mammals, including humans, contains cells that express nestin.Importantly, the distribution of nestin-positive cells in the pancreasdoes not correspond to the distribution of hormone-producing cells. Forexample, fluorescently labeled antibodies specifically reactive toinsulin or glucagon label the beta and alpha cells of the islets,respectively, whereas the inventors have observed that in mice, rats,and humans, fluorescently labeled nestin antibodies localize only tocertain cells of the ductular epithelium and not to alpha, beta, delta,and pancreatic peptide producing cells in pancreatic islets (FIG. 1).The inventors also observed that antibodies specific for collagen IV, amarker for vascular endothelial cells, galanin, a marker of nerveendings, and cytokeratin 19, a marker for ductal cells, do notcolocalize with nestin antibodies. Furthermore, the inventors have foundthat nestin-positive islet cells do not co-label with antibodies forinsulin, glucagon, somatostatin, or pancreatic polypeptide (FIG. 1).This suggests that these nestin-containing cells are not endocrinecells, ductal cells, neural cells, or vascular endothelial cells, butmay represent a truly distinct cell type within the islet that has notpreviously been described. The inventors have found nestin-positivecells in the islets, as well as localized regions of the pancreaticducts, and within centroacinar regions of the exocrine pancreas.

The expression of nestin mRNA in isolated islets was detected usingRT-PCR with RNA from isolated rat islets (FIG. 2). The functionalproperties of nestin-positive pancreatic cells were investigated usingcell culture techniques and by the isolation of nestin-positive cellsfrom islets, both of which are described below.

The inventors have also discovered that the liver of rats contains cellsthat express nestin (FIG. 13).

ATP-Binding Cassette (ABC) Multi-Drug Resistance Transporters (ABCG2(Brcp1) and MDR-) as Markers of Pancreatic Stem Cells.

Human islet-derived NIPs contain a substantial subpopulation of SP cellsthat co-express ABCG2, MDR1 and nestin: ABCG2 expression defines theside population (SP) phenotype of pluripotential stem cells in the bonemarrow, by virtue of excluding the dye Hoechst 33342. MDR-1 renders SPcells the capacity to replicate. SP cells (CD34 low/negative) do notproliferate and therefore cannot be expanded in vitro. The forcedexpression of MDR-1 in CD34 low negative SP cells causes them toproliferate (Bunting et al., 2000, Blood 96:902). NIPs are also CD34low/negative. Therefore NIPs are now defined as SP cells by virtue oftheir expression of ABCG2 and unlike marrow-derived SP cells, NIPsexpress MDR-1 and therefore can proliferate making it feasible to expandNIP SP cells ex vivo for purposes of transplantation.

Oct3/4 as a Marker of Pancreatic Stem Cells

NIPs express Oct3/4. Oct3/4 is a transcription factor belonging to thefamily of Pou homeodomain proteins (Niwa et al., 2002, Mol. Cell. Biol.22:1526; Niwa et al., 2000, Nat. Genet. 24:328; Niwa, 2001, Cell StructFunct 26:137; Shimazaki et al., 1993, EMBO 12:4489; Wang et al., 1996,Biochem Cell Biol 74:579; Nichols et al., 1998, Cell 95:379. This is animportant finding because the expression of Oct3/4 is strictlyrestricted to stem/progenitor cells (Niwa et al. 2002, supra; Niwa etal., 2000, supra; Niwa, 2001, supra). NIPs robustly express Oct3/4,thereby defining them per se to be stem/progenitor cells. That is ifOct3/4 expression is absolutely restricted to stem/progenitor cells, andOct3/4 is expressed in NIPs, then NIPs are stem/progenitor cells.

Integrin Subunits α6 and B1 as a Marker of Pancreatic Stem Cells

NIPs express the integrin subunits α and B1. α6/B1 integrin is thelaminin receptor (Giancotti and Ruoslahti, 1999, Science 285:1028). Ithas recently been shown that ES cells can be propagated in culturedishes coated with laminin and do not need to be grown on feeder celllayers (Xu et al., Nature Biotechnology, October 2001, 19:971). The cellsurface expressed integrins are essential for the maintenance of cellgrowth and development (see Giancotti et al., supra). The α6/B1 receptoris specific for recognition of laminin, which is an essential proteinexpressed in the blastocyst. Therefore α6/B1 is a marker of ES cells(stem/progenitor cells). NIPs express the integrin subunits α6 and B1.Therefore NIPs are similar to ES cells in this regard.

Hes-1 as a Marker of Pancreatic Stem Cells

The delta/Notch signaling pathway of lateral inhibition is critical forembryonic development. Much as been written about the importance ofNotch signaling. Notch signaling is also important in NIPs. Notchsignaling is absolutely required for the development of the pancreas(Apelquist et al., 1999, Nature 400:6747; Lammert et al., 2000, MechDev. 94; Jensen et al., 2000, Nat. Genet., 24:36; Jansen et al. Diabetes2000, 49:163). Ngn-3 is the key bHLH transcription factor determinantrequired for the development of the endocrine pancreas lineage. Hes-1 isa powerful suppressor of Ngn-3 expression. NIPs robustly express Hes-1.Therefore NIPs are progenitor cells prevented from differentiating intoendocrine cells. Therefore a method to block the expression of Hes-1 inthe NIPs will lead to their differentiation into endocrine cells(β-cells). Such a method for blocking Hes-1 expression could be the useof transfected antisense RNA, either by using small interfering RNAoligonucleotides or an expression plasmid expressing antisense Hes-1RNA.

GLP-1R as a Marker of Pancreatic Stem Cells

The inventors have further discovered that the pancreas of adultmammals, including humans, contains cell that express the glucagon likepeptide-1 receptor (GLP-1R). The invention is further based on thediscovery that GLP-1R-positive cells can co-localize withnestin-positive cells. The invention is also based on the conversediscovery; that nestin-positive pancreatic stem cells can co-localizewith GLP-1R-positive cells. The glucagon gene encodes a multifunctionalproglucagon, that is differentially process by pro-hormone convertasesin the pancreas and intestine to yield glucagon and the glucagon-likepeptides (GLP). GLP-1 has been shown to bind specifically to a G-proteincoupled receptor found in pancreatic β-cells to stimulate insulinsecretion via a cAMP-dependent pathway (Kiefer and Habener, 1999,Endocr. Rev. 20: 876; Drucker, 1998 Diabetes, 47: 159). The presentinvention is based, in part, on the discovery that the nestin-positivestem cells found in the pancreatic islets and ducts, as described above,also are GLP-1R-positive.

Nestin-positive-Islet-Progenitor cells (NIPs) were isolated from humanislet tissue and reacted with rabbit polyclonal antisera to the GLP-1R(Heller et al., supra), following the general immunocytochemicalprocedures outlined above. Receptor immunoreactivity was detected ingreater than 60% of NIPs examined (FIG. 22A). To further confirm theimmunocytochemical identification of GLP-1R in NIPs, RT-PCR wasperformed on mRNA prepared from NIP cells. Amplification of NIP mRNAwith the amplification primers 5′ gtgtggcggccaattactac 3′ (Forward); 5′cttggcaagtctgcatttga 3′ (Reverse) produced the expected 346 bp product(FIG. 22B) indicating that, in addition to expressing the GLP-1Rprotein, NIPs have the biosynthetic ability to produce GLP-1R. GLP-1R,therefore, in addition to nestin, is useful in the present invention asa marker for pancreatic stem cells.

Cytokeratin-19 as a Marker for a Distinct Population of Duct EpithelialCells

Cytokeratin-19 (CK-19) is another intermediate filament protein. CK-19and related cytokeratins have previously been found to be expressed inpancreatic ductal cells (Bouwens et al., 1994). The inventors havediscovered, however, that while CK-19 expression is indeed confined tothe ductules, fluorescent antibodies specific for CK-19 label distinctductal cells from those labeled with nestin-specific antibodies. Thissuggests that nestin-positive cells in islets may be a distinct celltype of ductal cell from CK-19 positive cells.

NIPs are CD34 Low/Negative:

The SP fraction of NIPs (left lower gate of FIG. 19B) express low tonull levels of CD34. This finding is important because CD34 low negativemarrow-derived SP cells, as contrasted to CD34-positive SP cells, arehighly pluripotential (see Goodell, 1999, Blood, 94:12545).Marrow-derived CD34 low/negative SP cells have a limited, if any,capacity to proliferate (Goodell et al., 1996, J. Exp. Med., 183:1797;Goodell et al., 1997 Nature Medicine, 3:1337). CD34 low/negative NIPs doproliferate in vitro, because they express Mdr-1.

Additional Markers of Pancreatic Stem Cells

The invention also provides for pancreatic stem cells that are positivefor at least one of the markers selected from the group consisting oflatrophilin (type 2) (Sudhof, 2001, Annu Rev Neurosci 24:933), C-kit(CD117) (Gibson et al., 2002, Adv Anat Pathol 9:65), SSTR-2, 3 and 4(somatostatin receptors) (Schulz et al., 2000, J Physiol Paris 94:259),SUR-1 (sulfonylurea receptor) (Winarto et al., 2001, Arch Histol Cytol64:59; Landgraf, 2000, Drugs Aging, 17:411), and Kir 6.2 (inwardrectifying K+ channel subunit with SUR-1 (Winarto et al., supra;Landgraf, supra).

The invention also provides for pancreatic stem cells that are negativefor at least one of CD45 (Sasaki et al., 2001, Int J Biochem Cell Biol33:1041), CD133 (Kobari et al., 2001, J Hematother Stem Cell Res10:273), MRC class II and MHC class II.

Isolated Stem Cells from Pancreatic Islets and Their Use

Stem cells can be isolated from a preparation of pancreatic tissue, forexample, islets obtained from a biopsy sample of tissue from a diabeticpatient. The stem cells can then be expanded ex vivo and the resultingcells transplanted back into the donor as an isograft. Inside the donor,they may differentiate to provide insulin-secreting cells such as betacells to replace beta cells lost to the autoimmune attack which causedthe diabetes. This approach can overcome the problems of immunerejection resulting from transplantation of tissue, for example, isletsfrom another individual who might serve as the donor. In one embodimentof the invention, the use of isografted stem cells allows anothertechnique to be performed in an effort to avoid the immune rejection,namely genetic therapy of the transplanted cells to render themresistant to immune attack, such as the autoimmunity present inindividuals with type 1 diabetes. A further advantage of using stemcells over whole islets is that transplanted stem cells candifferentiate in situ and better adapt to the host environment, forexample, providing appropriate microcirculation and a complement ofdifferent islet cell types which responds to the physiological needs ofthe host. Another embodiment of the invention contemplates the use ofpartially differentiated stem cells ex vivo, for example, to formprogenitor cells, which are subsequently transplanted into the host,with further differentiation optionally taking place within the host.Although the use of an isograft of stem cells, progenitor cells, orpseudo-islets is preferred, another embodiment contemplates the use ofan allograft of stem cells, progenitor cells, or pseudo-islets obtainedfrom another individual or from a mammal of another species.

In yet another embodiment of the invention, the stem cells areimmunologically blind or immunoprivileged. In one embodiment of thisaspect of the invention, immunoprivileged stem cells do not expresssufficient amounts of class I and/or class II major histocompatibilityantigens (a.k.a. HLA or human leukocyte antigen) to elicit an immuneresponse from the host. For example, these stem cells, obtained fromallogeneic or xenogeneic sources do not initiate a host versus graftresponse in immunocompetent transplant recipients.

In another embodiment of this aspect of the invention, immunoprivilegedstem cells do not express class I MHC antigens and/or class II MHCantigens. These stem cells, obtained from allogeneic or xenogeneicsources do not initiate a host versus graft response in immunocompetenttransplant recipients.

In another embodiment of the invention, human tissue grafts comprisingstem cells express both human specific class I and class II MHCantigens, but are recognized by immunocompetent mice as self, and do notundergo host versus graft rejection. These stem cells, obtained fromallogeneic or xenogeneic sources do not initiate a host versus graftresponse in immunocompetent transplant recipients.

The invention also provides for methods of isolating stem cells from axenogenic donor, and transplanting the resulting cells into a mammal ofanother species (e.g. murine stem cells are transplanted into a human,for example, a diabetic human patient) as a xenograft.

The invention provides for methods of performing isogeneic, allogeneicor xenogeneic transplants of nestin-positive stem cells wherein the stemcells are cultured for a period of time, for example, 2-4 hours, 4-5hours, 5-10 hours or 1-3 days prior to transplantation. The inventionalso provides for methods of performing isogeneic, allogeneic orxenogeneic transplants of a stem cell that is positive for at least oneof ABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin,Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2, SST-R3, SST-R4,SUR-1, Kir 6.2 and/or does not express one or all of CD34, CD45, CD133,MHC class I and MHC class II wherein the stem cells are cultured for aperiod of time, for example, 2-4 hours, 4-5 hours, 5-10 hours or 1-3days prior to transplantation.

The invention also provides for methods of performing isogeneic,allogeneic or xenogeneic transplants of nestin-positive stem cellswherein the stem cell is expanded for a period of time, for example, 2-4hours, 4-5 hours, 5-10 hours or 1-3 days prior to transplantation togive rise by cell division to other stem cells or progenitor cells.

The invention also provides for methods of performing isogeneic,allogeneic or xenogeneic transplants of a stem cell that is positive forat least one of ABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2),Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2,SST-R3, SST-R4, SUR-1, Kir 6.2 and/or does not express one or all ofCD34, CD45, CD133, MHC class I and MHC class II wherein the stem cell isexpanded for a period of time, for example, 2-4 hours, 4-5 hours, 5-10hours or 1-3 days prior to transplantation.

The invention also provides for methods of performing isogeneic,allogeneic or xenogeneic transplants of stem cells wherein thenestin-positive stem cells are induced to differentiate into aprogenitor cell by treatment with an agent selected from the groupconsisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, IDX-1, a nucleicacid molecule encoding IDX-1, GLP-1, exendin-4, betacellulin, activin A,TGF-β, and combinations thereof for a period of time, for example, 2-4hours, 4-5 hours, 5-10 hours or 1-3 days prior to transplantation.

In the case of a pancreatic stem cell, the stem cell subsequentlydifferentiates into a pancreatic progenitor cell.

The invention also provides for methods of performing isogeneic,allogeneic or xenogeneic transplants of a stem cell that is positive forat least one of ABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2),Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2,SST-R3, SST-R4, SUR-1, Kir 6.2 and/or does not express one or all ofCD34, CD45, CD133, MHC class I and MHC class II wherein the stem cellsare induced to differentiate into a progenitor cell by treatment with anagent selected from the group consisting of EGF, bFGF-2, high glucose,KGF, HGF/SF, IDX-1, a nucleic acid molecule encoding IDX-1, GLP-1,exendin-4, betacellulin, activin A, TGF-β, and combinations thereof fora period of time, for example, 2-4 hours, 4-5 hours, 5-10 hours or 1-3days prior to transplantation. In the case of a pancreatic stem cell,the stem cell subsequently differentiates into a pancreatic progenitorcell.

The invention provides for methods of performing isogeneic, allogeneicor xenogeneic transplants wherein nestin-positive stem cells are notcultured, expanded or differentiated prior to transplantation or whereinnestin-positive stem cells are cultured and/or expanded and/ordifferentiated prior to transplantation.

The invention also provides for methods of performing isogeneic,allogeneic or xenogeneic transplants wherein a stem cell that ispositive for at least one of ABCG2, Oct3/4, GLP-1 receptor, latrophilin(type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1,SST-R2, SST-R3, SST-R4, SUR-1, Kir 6.2 and/or does not express one orall of CD34, CD45, CD133, MHC class I and MHC class II are not cultured,expanded or differentiated prior to transplantation or wherein a stemcell that is positive for at least one of ABCG2, Oct3/4, GLP-1 receptor,latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit,MDR-1, SST-R2, SST-R3, SST-R4, SUR-1, Kir 6.2 and/or does not expressone or all of CD34, CD45, CD133, MHC class I and MHC class II iscultured and/or expanded and/or differentiated prior to transplantation.

Nestin-positive cells can be proliferated in culture from isolatedpancreatic islets and subsequently isolated to form a stem cell linecapable of essentially unlimited propagation.

In another embodiment, a stem cell that is positive for at least one ofABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin,Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2, SST-R3, SST-R4,SUR-1, Kir 6.2 and/or does not express one or all of CD34, CD45, CD133,MHC class I and MHC class II can be proliferated in culture fromisolated pancreatic islets and subsequently isolated to form a stem cellline capable of essentially unlimited propagation.

The inventors discovered that nestin expressing cells grow out ofcultured islets and can be observed growing around the islets as earlyas about four days in culture. These cells have a neuron-like morphology(FIG. 3), show nestin-positive staining, and express nestin mRNA. Isletscontaining nestin-positive cells can be separated from other cells,e.g., fibroblasts, that proliferate from the cultured islets by exposinga suspension of the islets to concanavalin A. The islets containingnestin-positive stem cells will not adhere to a concanavalin A coatedculture vessel, for example, allowing the islets to be simply decantedwhile other cell types remain attached to the vessel. The islets arethen plated on wells that do not have a concanavalin A coating, wherethey adhere. The details of the culture and isolation procedure aredescribed for rat cells in Example 1 below. Similar results have beenobtained with human cells.

Formation of Pseudo-Islets and Ductal Structures in Culture

One embodiment of the invention provides an alternative totransplantation of stem cells or progenitor cells by causing them toform pseudo-islet like aggregates that can be transplanted into apatient with insufficient islet cell mass to maintain physiologicalcontrol without hormone therapy. Islet-derived stem cells can beprepared from cultured islets as indicated above or obtained from apropagating stem cell line. The stem cells can then be induced todifferentiate by exposing them to various growth factors. This processis illustrated in Examples 2 and 3.

Differentiation of Stem Cells or Progenitor Cells to Islet Cells

Growth factors that may induce differentiation of pancreatic stem cellsinclude but are not limited to EGF-2, basic FGF, high glucose, KGF,HGF/SF, GLP-1, exendin-4, betacellulin, activin A, TGF-β, andcombinations thereof. GLP-1 refers to glucagon-like peptide-1. Highglucose refers to a higher glucose concentration than the concentrationnormally used in culturing the stem cells. For example, the stem cellscan be normally cultured and propagated in about 5.6 mM glucose, and thehigh glucose concentration refers to another concentration above 5.6 mM.In the preferred embodiment, a concentration of 16.7 mM is contemplated.In Example 2, one possible growth factor treatment is described usingbasic fibroblast growth factor (bFGF) and epidermal growth factor (EGF).

In addition to growth factors added to the medium of cultured cells,further growth factors can contribute to differentiation when stem cellsare implanted into an animal or a human. In that situation, many growthfactors-which are either known or unknown may be secreted by endogenouscells and exposed to the stem cells in situ. Implanted stem cells can beinduced to differentiate by any combination of endogenous andexogenously administered growth factors that are compatible with thedesired outcome, i.e., the final differentiated cell type or types andthe anatomical structures (e.g., tissues or organs) formed.

One embodiment provides an approach to stimulating differentiation, thatis to administer downstream effectors of growth factors or to transfectstem cells or progenitor cells with a nucleic acid molecule encodingsuch effectors. One example is IDX-1, which is a transcription factorinduced by GLP-1 or exendin-4. Introducing effectors such as IDX-1 cantrigger differentiation to form endocrine islet cells.

Analysis of Graft Rejection

The invention provides for an in vivo procedure for evaluating thesurvival of transplanted material. Experimental transplant rejection isanalyzed by transplanting an immunosuppressed or a non-immunosuppressedmammal, with a stem cell or a pseudo-islet like aggregate according tothe invention.

For example, non-immunosuppressed C57BL/6 mice are transplanted (forexample, under the renal capsules) with human stem cells according tothe invention. Graft rejection is analyzed by sacrificing the transplantrecipient and staining for viability, or performing immunocytochemicalstaining at the site of the grafted material (i.e., an organ or tissuepresent at the site of the grafted material) at a suitablepost-transplantation time point. The time point at which staining (forexample hematoxylin/eosin or immunostaining) of the site of the graftedmaterial is made can vary, for example, according to the averagesurvival time, or the expected survival time of a transplanted mammal.The site of the graft is analyzed, for example by staining, 1 day to 10years (i.e., 1, 5, 10, 30, 100 or more days, 1, 2, 5, or 10 years)post-transplantation, preferably 10 days to 1 year post-transplantationand most preferably, 10-100 days post-transplantation. For example, iftransplanted material is introduced under the renal capsule of a mouse,the kidney of the transplanted mouse is inspected. Transplanted materialis successfully engrafted (i.e., not rejected) if, the transplantedmaterial is still detectable and/or the transplanted material hasproliferated into a tissue mass.

Detection of the transplanted material and proliferation of thetransplanted material is determined, for example, by hematoxylin/eosinstaining of a frozen section prepared from the transplant site (i.e.,the kidney) and the detection of new growth that is not derived from thetransplant recipient (i.e., not host kidney derived). In the case ofxenogeneic transplantation, transplanted material is successfullyengrafted if specific immunostaining with antisera specific for anantigen from the species from which the transplanted material isderived, according to methods of immunocytochemical staining known inthe art and described herein, identifies positive cells. Alternatively,in embodiments wherein a xenogeneic transplantation is performed,transplanted material is successfully engrafted if molecules (i.e., aprotein or an antigen) derived from the transplant species (that is thespecies from which the transplanted material is derived) are detected inthe blood of the transplant recipient.

As used herein, “rejection” refers to rejection of transplanted materialby the immune system of the host. In one embodiment, “rejection means anoccurrence of more than 90% cell or tissue necrosis of the transplantedmaterial in response to the immune response of the host. In anotherembodiment, “rejection” means a decrease in the viability such that theviability of the transplanted material is decreased by 90% or more ascompared to the viability of the transplanted material prior totransplantation, in response to the immune response of the host. Adecrease in viability can be determined by methods well known in theart, including but not limited to trypan blue exclusion staining. Inanother embodiment, “rejection” means failure of the transplantedmaterial to proliferate. Proliferation can be measured by methods knownin the art including but not limited to hematoxylin/eosin staining. Theoccurrence of transplant rejection and/or the speed at which rejectionoccurs following transplantation will vary depending on factors,including but not limited to the transplanted material (i.e., the celltype, or the cell number) or the host (i.e., whether or not the host isimmunotolerant and/or has been treated with an immunosuppressive agent.

Methods of Transplantation

The invention provides for methods of transplantation in to a mammal. Astem cell, progenitor cell, or differentiated cell is “transplanted” or“introduced” into a mammal when it is transferred from a culture vesselinto a patient.

Transplantation, according to the invention can include the steps ofisolating a stem cell according to the invention and transferring thestem cell into a mammal or a patient. Transplantation according to theinvention can involve transferring a stem cell into a mammal or apatient by injection of a cell suspension into the mammal or patient,surgical implantation of a cell mass into a tissue or organ of themammal or patient, or perfusion of a tissue or organ with a cellsuspension. The route of transferring the stem cell or transplantation,will be determined by the need for the cell to reside in a particulartissue or organ and by the ability of the cell to find and be retainedby the desired target tissue or organ. In the case where a transplantedcell is to reside in a particular location, it can be surgically placedinto a tissue or organ or simply injected into the bloodstream if thecell has the capability to migrate to the desired target organ.

Transplantation, according to the invention, can include the steps ofisolating a stem cell according to the invention, and culturing andtransferring the stem cell into a mammal or a patient. In anotherembodiment, transplantation, as used herein, can include the steps ofisolating a stem cell according to the invention, differentiating thestem cell, and transferring the stem cell into a mammal or a patient.Transplantation, as used herein, can include the steps of isolating astem cell according to the invention, differentiating and expanding thestem cell and transferring the stem cell into a mammal or a patient.

Methods of Treating Insulin-Dependent Diabetes Using Pancreatic StemCells

Stem cells are useful to replace lost beta cells from Type 1 diabetespatients or to increase the overall numbers of beta cells in Type 2diabetes patients. The diabetes patient will preferably serve as thedonor of pancreatic tissue used to produce stem cells, progenitor cells,or pseudo-islet like aggregates. Stem cells exist within the adultpancreatic islets as well as the pancreatic ducts. After a diabeticpatient undergoes pancreatic biopsy, islets are isolated from the biopsytissue and prepared for culture ex vivo preferably within 24 hours. Stemcells can be proliferated and isolated by the methods described abovewithin 2-3 weeks. Stem cells can be transplanted back into the patientdirectly following isolation or after a period of differentiation whichis induced by growth factors. Islets can be produced by subculture asdescribed in Example 2. The whole process of surgical pancreas biopsyand transplantation can be performed within a period of about 30 days.

In one embodiment of the invention, pluripotential stem cells are used.These cells are immunologically blinded or immunoloprivileged, such thatin allogeneic or xenogeneic transplants, they are recognized as self bythe recipient, and are not MHC restricted by class I or class IIantigens. In one aspect of this embodiment of the invention, these cellsdo not express MHC class I and/or class II antigens.

In another embodiment of the invention, the recipient of the transplantmay demonstrate host vs. graft rejection of other transplanted cells,which can be combated by the administration of blocking antibodies to,for example, an autoantigen such as GAD65, by the administration of oneor more immunosuppressive drugs described herein, or by any method knownin the art to prevent or ameliorate autoimmune rejection.

Alternatively, stem cells isolated from a non-human mammal according tothe invention, are transplanted into a human diabetes patient. Prior tothe transplantation step the stem cells may be cultured, and/or expandedand/or differentiated.

Methods of Treating Patients Suffering from Liver Disease UsingPancreatic Stem Cells

The ability of pancreatic stem or progenitor cells to transdifferentiateto form hepatocytes is well known (Bisgaard & Thorgeirsson, 1991). Thepancreatic stem cells of the present invention can be used to providehepatocytes for a patient suffering from a liver disease such ascirrosis, hepatitis, or hepatic cancer in which the functional mass ofhepatic tissue has been reduced. The stem cells of the invention canalso be treated by gene therapy to correct a genetic defect andintroduced into a patient to restore hepatic function. Nestin-positivestem cells can be differentiated either in culture or in vivo byapplying one or more growth factors, or other treatment such astransfection with a nucleic acid molecule, that results indifferentiation of the stem cells to hepatocytes. In one embodiment, theinvention contemplates the use of cyclopamine to suppress, for example,sonic hedgehog, resulting in hepatocyte formation. In another embodimentof the invention, the stem cells can be transplanted without any ex vivotreatment and the appropriate growth factors can be provided in situwithin the patient's body. In yet another embodiment, the stem calls canbe treated with growth factors or other agents ex vivo and subsequentlytransplanted into the patient in a partially differentiated orterminally differentiated state. Other aspects of the invention,including methods of transfecting stem cells or progenitor cells,dosages and routes of administration, pharmaceutical compositions,donor-isograft protocols, and immunosuppression methods, can bepracticed with transdifferentiation to hepatocytes just as for thedifferentiation to pancreatic tissues.

The invention specifically contemplates transplanting into patientsisogeneic, allogeneic, or xenogeneic stem cells, or any combinationthereof.

Methods of Stem Cell Transfection

A variety of methods are available for gene transfer into pancreaticstem cells. Calcium phosphate precipitated DNA has been used butprovides a low efficiency of transformation, especially for nonadherentcells. In addition, calcium phosphate precipitated DNA methods oftenresult in insertion of multiple tandem repeats, increasing thelikelihood of disrupting gene function of either endogenous or exogenousDNA (Boggs, 1990). The use of cationic lipids, e.g., in the form ofliposomes, is also an effective method of packaging DNA for transfectingeukaryotic cells, and several commercial preparations of cationic lipidsare available. Electroporation provides improved transformationefficiency over the calcium phosphate protocol. It has the advantage ofproviding a single copy insert at a single site in the genome. Directmicroinjection of DNA into the nucleus of cells is yet another method ofgene transfer. It has been shown to provide efficiencies of nearly 100%for short-term transfection, and 20% for stable DNA integration.Microinjection bypasses the sometimes problematic cellular transport ofexogenous DNA through the cytoplasm. The protocol requires small volumesof materials. It allows for the introduction of known amounts of DNA percell. The ability to obtain a virtually pure population of stem cellswould improve the feasibility of the microinjection approach to targetedgene modification of pancreatic stem cells. Microinjection is a tedious,highly specialized protocol, however. The very nature of the protocollimits the number of cells that can be injected at any given time,making its use in large scale production limited. Gene insertion intopancreatic stem cells using retroviral methods is the preferred method.Retroviruses provide a random, single-copy, single-site insert at veryhigh transfection efficiencies. Other such transfection methods areknown to one skilled in the art and are considered to be within thescope of this invention.

Retroviral Transformation of Pancreatic Stem Cells

Gene transfer protocols for pancreatic cells can involve retroviralvectors as the “helper virus” (i.e., encapsidation-defective viralgenomes which carry the foreign gene of interest but is unable to formcomplete viral particles). Other carriers such as DNA mediated transfer,adenovirus, SV40, adeno-associated virus, and herpes simplex virusvectors can also be employed. Several factors should be considered whenselecting the appropriate vector for infection. It is sometimespreferable to use a viral long terminal repeat or a strong internalpromoter to express the foreign gene rather than rely on splicedsubgenomic RNA.

The two primary methods of stem cell transformation are co-culture andsupernatant infection. Supernatant infection involves repeated exposureof stem cells to the viral supernatant. Co-culture involves thecommingling of stem cells and an infected “package cell line” (seebelow) for periods of 24 to 48 hours. Co-culture is typically moreefficient than supernatant infection for stem cell transformation. Afterco-culture, infected stem cells are often further cultured to establisha long term culture (LTC).

The cell line containing the helper virus is referred to as the packagecell line. A variety of package cell lines are currently available. Animportant feature of the package cell line is that it does not producereplication-competent helper virus.

In one embodiment of the invention animals or patients from whom stemcells are harvested may be treated with 5-fluorouracil (5-FU) prior toextraction. 5-FU treated stem cells are more susceptible to retroviralinfection than untreated cells. 5-FU stem cells dramatically reduce thenumber of clonogenic progenitors, however.

In another embodiment, harvested stem cells may be exposed to variousgrowth factors, such as those employed to promote proliferation ordifferentiation of pancreatic stem cells. Growth factors can beintroduced in culture before, during, or after infection to improve cellreplication and transduction. Studies report the use of growth factorsincrease transformation efficiency from 30 to 80%.

Typical Retroviral Transformation Protocol

The ex vivo transduction of mammalian pancreatic stem cells andsubsequent transplantation into nonablated recipients sufficient toobtain significant engraftment and gene expression in various tissuescontaining their progeny cells has been shown in mice. The target cellsare cultured for 2-4 days in the presence of a suitable vectorcontaining the gene of interest, before being injected in to therecipient.

Specifically, bone marrow stem cells were harvested from male donor (4-8weeks old) BALB/c AnNCr mice (National Cancer Institute, Division ofCancer Treatment Animal Program, Frederick, Md.). The cells were platedat a density of 1-2×10⁷ cells/10 cm dish and cultured for 48 hours inDulbecco's modified Eagle's medium (DMEM) containing; 10%heat-inactivated fetal bovine serum, glutamine, Pen/Strep, 100 U/ml ofinterleukin-6 (IL-6) and stem cell factor (SCF; Immunex, Seattle, Wash.)to stimulate cell growth (Schiffmann, et. al., 1995).

Concurrently, a viral package cell line was cultured for 24 hours. Thepackage cell line used by Schiffmann, et al. was GP+E86 and the viralvector was the LG retroviral vector based on the LN series of retroviralvectors.

After the appropriate incubation period, 1-2×10⁷ stem cells were platedon a 10 cm dish containing the viral package cells and co-cultured for48 hours in the presence of 8 μg/ml of polybrene and under the samegrowth factor stimulation conditions as the donor stem cells. The stemcells were then harvested, washed of growth media and injected intorecipient mice at dosages of 2×10⁷ cells/injection for multipleinjections (total of 5 injections either daily or weekly).

Successful stem cell transduction and engraftment of stem cells can bedetermined through, for example, PCR analysis, immunocytochemicalstaining, Southern Northern or Western blotting, or by other suchtechniques known to one skilled in the art.

Mammals

Mammals that are useful according to the invention include any mammal(for example, human, mouse, rat, sheep, rabbit, goat, monkey, horse,hamster, pig or cow). A non-human mammal according to the invention isany mammal that is not a human, including but not limited to a mouse,rat, sheep, rabbit, goat, monkey, horse, hamster, pig or a cow.

Dosage and Mode of Administration

By way of example, a patient in need of pancreatic stem cells asdescribed herein can be treated as follows. Cells of the invention canbe administered to the patient, preferably in a biologically compatiblesolution or a pharmaceutically acceptable delivery vehicle, byingestion, injection, inhalation or any number of other methods. Apreferred method is endoscopic retrograde injection. Another preferredmethod is injection into the pancreatic artery. Another preferred methodis injection or placement of the cells or pseudo-islet like aggregatesinto the space under the renal capsule. The dosages administered willvary from patient to patient; a “therapeutically effective dose” can bedetermined, for example but not limited to, by the level of enhancementof function (e.g., insulin production or plasma glucose levels).Monitoring levels of stem cell introduction, the level of expression ofcertain genes affected by such transfer, and/or the presence or levelsof the encoded product will also enable one skilled in the art to selectand adjust the dosages administered. Generally, a composition includingstem cells will be administered in a single dose in the range of 10⁵-10⁸cells per kg body weight, preferably in the range of 10⁶-10⁷ cells perkg body weight. This dosage may be repeated daily, weekly, monthly,yearly, or as considered appropriate by the treating physician. Theinvention provides that cell populations can also be removed from thepatient or otherwise provided, expanded ex vivo, transduced with aplasmid containing a therapeutic gene if desired, and then reintroducedinto the patient.

Pharmaceutical Compositions

The invention provides for compositions comprising a stem cell accordingto the invention admixed with a physiologically compatible carrier. Asused herein, “physiologically compatible carrier” refers to aphysiologically acceptable diluent such as water, phosphate bufferedsaline, or saline, and further may include an adjuvant. Adjuvants suchas incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide,or alum are materials well known in the art.

The invention also provides for pharmaceutical compositions. In additionto the active ingredients, these pharmaceutical compositions may containsuitable pharmaceutically acceptable carrier preparations which can beused pharmaceutically.

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, foringestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethyl cellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hank'ssolution, Ringer' solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Additionally, suspensions of the active solventsor vehicles include fatty oils such as sesame oil, or synthetic fattyacid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

For nasal administration, penetrants appropriate to the particularbarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner known in the art, e.g. by means of conventionalmixing, dissolving, granulating, dragee-making, levitating, emulsifying,encapsulating, entrapping or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc . . . . Saltstend to be more soluble in aqueous or other protonic solvents that arethe corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2%sucrose, 2%-7% mannitol at a Ph range of 4.5 to 5.5 that is combinedwith buffer prior to use.

After pharmaceutical compositions comprising a compound of the inventionformulated in a acceptable carrier have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition with information including amount, frequency andmethod of administration.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples, which are provided herein for purposes ofillustration only and are not intended to limit the scope of theinvention.

EXAMPLE 1

Isolation of Nestin-Positive Stem Cells from Rat Pancreas.

Rat islets were isolated from the pancreata of 2-3 month oldSprague-Dawley rats using the collagenase digestion method described byLacy and Kostianovsky. Human islets were provided by the DiabetesResearch Institute, Miami, Fla. using collagenase digestion. The isletswere cultured for 96 hrs at 37° C. in 12-well plates (Falcon 3043plates, Becton Dickinson, Lincoln Park, N.J.) that had been coated withconcanavalin A. The culture medium was RPMI 1640 supplemented with 10%fetal bovine serum, 1 mM sodium pyruvate, 10 mM HEPES buffer, 100 μg/mlstreptomycin, 100 units/ml penicillin, 0.25 μg/ml amphotericin B (GIBCOBRL, Life Science Technology, Gaithersburg, Md.), and 71.5 mMβ-mercaptoethanol (Sigma, St. Louis, Mo.).

After 96 hrs, fibroblasts and other non-islet cells had adhered to thesurface of concanavalin A coated wells and the islets remained floating(did not adhere to the surface). At this time, the media containing theislets were removed, centrifuged down, and the purged islets replated in12-well plates without a coating of concanavalin A. The islets were thencultured in the above RPMI 1640 medium supplemented with 20 ng/ml ofbasic fibroblast growth factor-2 and 20 ng/ml of epidermal growthfactor.

The islets adhered to the surface of the plates, and cells grew out andaway from the islets in a monolayer. These cells that form a monolayerwere nestin-positive by immunostaining with a rabbit anti-rat nestinantiserum developed by Dr. Mario Vallejo at the Massachusetts GeneralHospital. Other nestin antibodies may be used, for example the R.401antibody described hereinabove, or the MAB533 antibody. A monoclonalantibody specific for rat embryo spinal cord nestin, MAB353, ATCC No.1023889; is described in Journal of Neuroscience 1996; 16:1901-100; andalso available from Chemicon International, Single Oak Dr., Temecula,Calif. 92590 USA. After two weeks of culture, several (3-5) of thenestin-positive monolayer cells were removed by picking with a capillarytube (cylinder cloning) and were replated on the 12-well plates (notcoated with concanavalin A) and cultured in the RPMI 1640 medium furthersupplemented with bFGF-2 and EGF. The cells propagated at a rapid rateand reached confluence after six days of culture. After 12 days ofculture, the cell monolayer formed waves in which they begin to pile upin a co-linear manner. On day 15 of culture, the cell waves began tocondense, migrate into spheroid bodies and by day 17 the surface of thewells contained these spheroid bodies (ca. 100 μm in diameter), emptyspaces, and a few areas of remaining monolayer cells. Several of thesemonolayer cells were re-picked and re-cloned and the process describedabove occurred again in precisely the same temporal sequence.

EXAMPLE 2

Differentiation of Pancreatic Stem Cells to Form Islet

Pancreatic islets from rats were first cultured in RPMI mediumcontaining 10% fetal bovine serum using concanavalin-A coated 12-wellplates. The islets were maintained in culture for three days in theabsence of added growth factors other than those supplied by fetalbovine serum. After this period, during which the islets did not attach,the islets were transferred to fresh plates without concanavalin A. Thestem cells were then stimulated to proliferate out from the islets as amonolayer by exposing them to bFGF-2 (20 ng/ml) and EGF (20 ng/ml) for24 days. After the 24 day period, the monolayer was confluent. Amongthem was a population of cells surrounding the islets. Cells from thatpopulation were picked and subcloned into new 12-well plates and againcultured in the medium containing bFGF and EGF. The subcloned cellsproliferated rapidly into a monolayer in a clonal fashion, expandingfrom the center to the periphery. The cells became confluent at day 6and then started to form a wave of overlapping cells on day 12. By day17 the cells migrated almost entirely into spherical structures andtubular structures resembling islet-like structures (pseudo-islet likeaggregates) and duct-like structures (pseudo-ducts) (FIG. 4). RT-PCRanalysis revealed that the pseudo-islet like aggregates were expressingNCAM (a marker for endocrine cells, see FIG. 5), cytokeratin 19 (amarker for ductal cells, see FIG. 5), and the transcription factorbrain-4 (a beta cell marker). Treatment with growth factors is requiredto achieve terminal differentiation to mature islet cells.

EXAMPLE 3

Isolation and Culture of Human or Rat Pancreatic Islets

Human pancreatic islets were isolated and cultured. Human islet tissuewas obtained from the islet distribution program of the Cell TransplantCenter, Diabetes Research Institute, University of Miami School ofMedicine and the Juvenile Diabetes Foundation Center for IsletTransplantation, Harvard Medical School, Boston, Mass. Thoroughly washedislets were handpicked, suspended in modified RPMI 1640 media (11.1 mMglucose) supplemented with 10% fetal bovine serum, 10 mM HEPES buffer, 1mM sodium pyruvate, 100 U per mL penicillin G sodium, 100 μg per mLstreptomycin sulfate, 0.25 ng per mL amphotericin B, and 71.5 μMβ-mercaptoethanol, and added to Falcon 3043 12-well tissue cultureplates that had been coated with Concanavalin A (ConA). The isletpreparation was incubated for 96 hrs at 37° C. with 95% air and 5% CO₂.In these conditions, many islets remained in suspension (floated),whereas fibroblasts and other non-islet cells attached to thesubstratum. After 96 h of incubation the media containing the suspendedislets was carefully removed, the islets were manually picked andresuspended in the modified RPMI 1640 media now further supplementedwith 20 ng/mL each of basic fibroblast growth factor (bFGF) andepidermal growth factor (EGF). The islet suspension (containing 20-30islets per well) was added to 12-well tissue culture plates not coatedwith ConA. The islets immediately adhered to the surfaces of the plates.Within several days, a monolayer of cells was observed growing out andaway from the islets. In certain instances, human-derived cells werecultured in modified RPMI media containing 2.5 mM glucose, and severalgrowth factor combinations that include activin-A (2 nM), hepatocytegrowth factor (100 pM), or betacellulin (500 pM). In those experimentsin which cells were challenged with 10 mM nicotinamide, the mediacontained no serum or growth factors.

EXAMPLE 4

Effects of Glucose and GLP-1 on Differentiation of Pancreatic StemCells.

Elevation of plasma glucose concentration leads to increased pancreaticislet size. The effect of the glucose concentration in the culturemedium was therefore investigated using isolated islets, which containnestin-positive stem cells. Rat pancreatic islets were cultured in amedium containing high (16.7 mM) glucose or in normal (5.6 mM) glucose.After four days, RT-PCR was performed to determine the level of nestinmRNA. The results indicated a three-fold stimulation of nestin mRNAlevels in the islets cultured in high glucose compared to the isletscultured in normal glucose (FIG. 6).

Similarly, injection of glucagon-like peptide-1 (GLP-1) into mice wasfound to increase islet mass by 2-fold in 48 hours. Knockout mice havinga disrupted gene for GLP-1 receptor were examined for nestin expressionin pancreatic islets. Immunostaining using a nexin antibody was found tobe markedly reduced compared to normal mice with GLP-1 receptors.

Animal Model of Diabetes Mellitus

Treatments for diabetes mellitus type that result in relief of itssymptoms are tested in an animal which exhibits symptoms of diabetes. Itis contemplated that the animal will serve as a model for agents andprocedures useful in treating diabetes in humans. Potential treatmentsfor diabetes can therefore be first examined in the animal model byadministering the potential treatment to the animal and observing theeffects, and comparing the treated animals to untreated controls.

The non-obese diabetic (NOD) mouse is an important model of type I orinsulin dependent diabetes mellitus and is a particularly relevant modelfor human diabetes (see Kikutano and Makino, 1992, Adv. Immunol. 52:285and references cited therein, herein incorporated by reference). Thedevelopment of type I diabetes in NOD mice occurs spontaneously andsuddenly without any external stimuli. As NOD mice develop diabetes,they undergo a progressive destruction of β-cells which is caused by achronic autoimmune disease. The development of insulin-dependentdiabetes mellitus in NOD mice can be divided roughly into two phases:initiation of autoimmune insulitis (lymphocytic inflammation in thepancreatic islets) and promotion of islet destruction and overtdiabetes. Diabetic NOD mice begin life with euglycemia, or normal bloodglucose levels, but by about 15 to 16 weeks of age the NOD mice startbecoming hyperglycemic, indicating the destruction of the majority oftheir pancreatic β-cells and the corresponding inability of the pancreasto produce sufficient insulin. In addition to insulin deficiency andhyperglycemia, diabetic NOD mice experience severe glycosuria,polydypsia, and polyuria, accompanied by a rapid weight loss. Thus, boththe cause and the progression of the disease are similar to humanpatients afflicted with insulin dependent diabetes mellitus. Spontaneousremission is rarely observed in NOD mice, and these diabetic animals diewithin 1 to 2 months after the onset of diabetes unless they receiveinsulin therapy.

The NOD mouse is used as an animal model to test the effectiveness ofthe various methods of treatment of diabetes by administering a stemcell preparation according to the invention. As such, treatment viaadministration of stem cells are tested in the NOD mouse for theireffect on type I diabetes.

The stem cells are administered to a NOD mouse, typicallyintraperitoneally, according to the following dosage amounts. NOD miceare administered about 1×10¹ to 1×10⁴ cells per mouse. Administration ofthe cells is started in the NOD mice at about 4 weeks of age, and iscontinued for 8 to 10 weeks, e.g., 3 times a week. The mice aremonitored for diabetes beginning at about 13 weeks of age, being testedtwice per week according to the methods described below. The effects oftreatment are determined by comparison of treated and untreated NODmice.

The effectiveness of the treatment methods of the invention on diabetesin the NOD mice is monitored by assaying for diabetes in the NOD mice bymeans known to those of skill in the art, for example, examining the NODmice for polydipsia, polyuria, glycosuria, hyperglycemia, and insulindeficiency, or weight loss. For instance, the level of urine glucose(glycosuria) can be monitored with Testape (Eli Lilly, Indianapolis,Ind.) and plasma glucose levels can be monitored with a Glucometer 3Blood Glucose Meter (Miles, Inc., Elkhart, Ind.) as described by Burkly,1999, U.S. Pat. No. 5,888,507, herein incorporated by reference.Monitoring urine glucose and plasma glucose levels by these methods, NODmice are considered diabetic after two consecutive urine positive testsgave Testape values of +1 or higher or plasma glucose levels >250 mg/dL(Burkly, 1999, supra). Another means of assaying diabetes in NOD mice isto examine pancreatic insulin levels in NOD mice. For example,pancreatic insulin levels can be examined by immunoassay and comparedamong treated and control mice (Yoon, U.S. Pat. No. 5,470,873, hereinincorporated by reference). In this case, insulin is extracted frommouse pancreas and its concentration is determined by itsimmunoreactivity, such as by radioimmunoassay techniques, using mouseinsulin as a standard.

In addition to monitoring NOD mice for diabetes in general, the effectsof the inventive methods of treatment are also monitored forgene-specific or gene product-specific effects if the stem cellsadministered were transformed or transfected with a heterologous gene,thereby allowing a correlation to be drawn between expression of theheterologous gene and its effects on diabetes. For example, the presenceof the heterologous gene product may be examined by immunohistochemistryof the pancreatic β-cells of NOD mice for the gene product and forinsulin. The expression of the patched and smoothened genes is furtherexamined in NOD mouse islets by detection of the RNA transcript for thepatched and smoothened receptors. Reverse transcription-polymerase chainreaction (RT-PCR) amplification is performed by known means to amplify afragment of mouse patched or smoothened cDNA, and analyzed by agarosegel electrophoresis, according to standard means. The identification ofthe amplified cDNA fragment is confirmed as corresponding to the patchedor smoothened RNA by hybridization of the amplified fragment with aradiolabeled internal oligonucleotide probe for the patched orsmoothened genes, or by other such methods as known to one skilled inthe art.

EXAMPLE 5

Immunocytochemical Identification of Nestin Positive Human and RatPancreatic Stem Cells

Pancreatic islets were analyzed for nestin expression. Islets and stemcells were isolated as described above. Nestin expression was observedby immunocytochemical staining in a distinct population of cells withindeveloping islet clusters of embryonic day 16 (E16) rat pancreas (FIG.8A) and in islets of the adult pancreas (postnatal 60 days) (FIG. 8B).Immunocytochemical staining was performed as follows.

Cryosections (6 μM) prepared from embryonic day 16 and adult (60 day)rat pancreata as well as cells were fixed with 4% paraformaldehyde inphosphate. Cells were first blocked with 3% normal donkey serum for 30min at room temperature and incubated with primary antisera overnight at4° C. The antisera were rinsed off with PBS and incubated with therespective Cy-3 and Cy-2 labeled secondary antisera for 1 hour at roomtemperature. Slides were then washed with PBS and coverslipped withfluorescent mounting medium (Kirkegaard and Perry Labs, Gaithersburg,Md.). Tissue sections were incubated overnight at 4° C. with primaryantisera. Primary antisera were then rinsed off with PBS, and slideswere blocked with 3% normal donkey serum for 10 min at room temperaturebefore incubation with donkey anti-Cy3 (indocarbocyanine) and eitheranti-guinea pig (insulin), anti-mouse (glucagon), or anti-sheep(somatostatin) sera DTAF (Jackson Immuno Research Laboratories, WestGrove, Pa.) for 30 min at room temperature. Slides were then rinsed withPBS and coverslipped with fluorescent mounting medium (Kirkegaard andPerry Laboratories, Gaithersburg, Md.). Fluorescence images wereobtained using a Zeiss Epifluorescence microscope equipped with anOptronics TEC-470 CCD camera (Optronics Engineering, Goleta, Calif.)interfaced with a PowerMac 7100 installed with IP Lab Spectrum analysissoftware (Signal Analytics Corp, Vienna, Va.).

The nestin-positive cells are distinct from β-, α-, δ-, and PP-cellsbecause they do not co-stain with antisera to the hormones insulin (FIG.8A & B), glucagon, somatostatin, or pancreatic polypeptide. Thenestin-positive cells also do not co-stain with antisera to collagen IV,a marker for vascular endothelial cells (FIG. 8C) nor with an antiserumto galanin, a marker for nerve cells or a monoclonal antibody tocytokeratin 19, a specific marker for ductal cells (FIG. 8).Nestin-positive staining is associated with distinct cells within theislets clearly observed by nuclear costaining (FIG. 4D).

EXAMPLE 6

Identification of Nestin Positive Human and Rat Stem Cells by RT-PCR

To confirm the immunocytochemical identification of nestin expression inpancreatic islets, we performed an RT-PCR of the nestin mRNA using totalRNA prepared from freshly isolated rat islets and human islet tissue.RT-PCR was performed according to the following method.

Total cellular RNA prepared from rat or human islets was reversetranscribed and amplified by PCR for 35 cycles as described previously(Daniel et al., 1998, Endocrinology, 139:3721-3729). Theoligonucleotides used as primers or amplimers for the PCR and as probesfor subsequent Southern blot hybridization are: Rat nestin: Forward,5′gcggggcggtgcgtgactac3′; Reverse, 5′aggcaagggggaagagaaggatgt3′;Hybridization, 5′aagctgaagccgaatttccttgggataccagagga3′. Rat Forward,5′acagccagtacttcaagacc3′; keratin 19: Reverse, 5′ctgtgtcagcacgcacgtta3′;Hybridization, 5′tggattccacaccaggcattgaccatgcca3′. Rat NCAM: Forward,5′cagcgttggagagtccaaat3′; Reverse, 5′ttaaactcctgtggggttgg3′;Hybridization, 5′aaaccagcagcggatctcagtggtgtggaacgatgat3′. Rat IDX-1Forward, 5′atcactggagcagggaagt3′ Reverse, 5′gctactacgtttcttatct3′Hybridization, 5′gcgtggaaaagccagtggg3′ Human Forward,5′agaggggaattcctggag3′; nestin: Reverse, 5′ctgaggaccaggactctcta3′;Hybridization, 5′tatgaacgggctggagcagtctgaggaaagt3′. Human Forward,5′cttttcgcgcgcccagcatt3′; keratin: Reverse, 5′gatcttcctgtccctcgagc3′;Hybridization, 5′aaccatgaggaggaaatcagtacgctgagg3′. Human Forward,5′atctggactccaggcgtgcc3′; glucagon: Reverse, 5′agcaatgaattccttggcag3′;Hybridization, 5′cacgatgaatttgagagacatgctgaaggg3′.

Primers were selected from two different exons and encompassed at leastone intronic sequence. In addition, an RT minus control was run for mostsamples. PCR cycling was at 94° C. for 1 min followed by 94° C. for 10secs, 58/56° C. for 10 secs, 72° C. for 1 min, 35 cycles, and 72° C. for2 min. The annealing temperature was 58° C. for rat nestin and 56° C.for the remaining primer pairs.

For Southern hybridization oligonucleotide probes were radiolabeled withT4 polynucleotide kinase and γ³²P ATP. Radiolabeled probes werehybridized to PCR products that had been transferred to nylon membranesat 37° C. for one hour, then washed in 1×SSC+0.5% SDS at 55° C. for10-20 min or 0.5×SCC+0.5% SDS at 42° for the human PCR products.

The RT-PCR generated products of the correctly predicted size (FIG. 8E,upper panels) and were confirmed by Southern blotting (FIG. 8E, lowerpanels) and by DNA sequencing of the products. These data demonstratethe identification of a new cell type in pancreatic islets thatexpresses nestin and may represent an islet pluripotential stem cellsimilar to the nestin-positive stem cells in the central nervous system.

EXAMPLE 7

The ATP-dependent Transporter ABCG2 is Expressed in Nestin-positiveCells Derived from Pancreatic Islets

Human islet-derived NIPs contain a substantial subpopulation of SP cellsthat co-express ABCG2, MDR1 and nestin. Nestin was first shown to be amarker of neural and muscle stem/progenitor cells (Lendahl, et al.,1990, Cell 60:585: Zimmerman et al.; 1994, Neuron 12:11). Neural stemcells especially exhibit a high degree of plasticity giving rise toneurons and different types of glial cells. Neural stem/progenitor cellsalso differentiate into hematopoietic cells, which would make them trulypluripotential stem cells (Shih et al., 2001, Blood 98:2412). The bestexamples of pluripotential adult stem cells to date are those derivedfrom the bone marrow, so called side population (SP) cells (Goodell etal., 1996, J. Exp Med 183:1797). SP cells have been identified by theirproperty to effectively exclude the fluorescent vital dye Hoechst 33342and contain the vast majority of bone marrow repopulating cells. SPcells represent 0.05% of the whole bone marrow of adult humans and mice,and apart from giving rise to all hematopoietic lineages they can alsogive rise to skeletal and cardiac muscle as well as to endothelial cells(Gussoni et al., 1999, Nature 401:390; Jackson et al., 2001, J ClinInvest 107:1395). The ATP-\binding cassette transporter ABCG2 (BCRP1)has been demonstrated to be a major component of the SP phenotype, thusoffering a molecular means to specifically identify SP-cells (Kim etal., 2002, Clin Cancer Res 8:22; Scharenberg et al., Blood 99:507; Zhouet al., 2001, Nat Med 7:1028). Notably, marrow-derived SP cells have alimited, if any, capacity to proliferate in vitro (Bunting et al., 2000,Blood 96:902; Storms et al., 2000, Blood 96:2125), but enforcedP-glycoprotein pump (MDR-1) function in bone marrow cells results in theexpansion of SP stem cells in vitro and repopulating cells in vivo(Bunting et al., 2002, Stem Cells 20:11).

NIPs, linked to neural stem cells by their expression of nestin, alsoshow characteristics of bone marrow SP stem cells, by virtue of theirexpression of ABCG2 in a substantial subpopulation of NIPs identified bythe Hoechst 33342 exclusion assay. This side population of NIPs alsoexpresses MDR-1, contributing to their continued expansion in vitro.Thus NIPs may be a potential source of adult pluripotentialstem/progenitor cells useful for the production of islet tissue fortransplantation into diabetic subjects.

Human pancreatic islets were obtained from the islet distributionprograms of the Cell Transplant Center, Diabetes Research Institute,University of Miami School of Medicine (Miami, Fla.), and the JuvenileDiabetes Research Foundation Center for Islet Transplantation, HarvardMedical School (Boston, Mass.). NIPs were isolated as previouslydescribed (Zulewski et al., 2001, Diabetes 50:521). Briefly, humanpancreatic islets were handpicked, incubated at 37° C. at 5% CO₂ inconcanavalin A (ConA) coated culture dishes in modified RPMI 1640 mediasupplemented with 10% fetal bovine serum, 10 mmol/l HEPES buffer, 1mmol/l sodium pyruvate, 71.5 μmol/l β-mercaptoethanol andantibiotic-antimycotic (Gibco Life Technologies, Gaithersburg, Md.).After 96 hours floating islets were transferred into fresh media furtherthat was supplemented with 20 ng/ml basic fibroblast growth factor(bFGF) and 20 ng/ml epidermal growth factor (EGF) (both from Sigma, St.Louis, Mo.) in new plastic culture dishes without ConA coating. Theislets attached to the uncoated plastic surface and within several daysa monolayer of cells was observed growing out and away from the islets.Cells from the periphery of the monolayer, nestin-positive islet-derivedprogenitor cells (NIPs), were transferred to a new culture dish andgrown to near confluence under the same conditions as above. Cells werefurther split into two culture dishes to perform Hoechst 33342 dyeexclusion assays with and without verapamil, an inhibitor of H33342 dyetransport (Goodell et al., 1996, supra).

RT-PCR demonstrated the expression of ABCG2 (FIG. 19A). TheRT-PCR-generated DNA product was sequenced and shown to be identical(586 bps) with that of the human ABCG2 (GenBank Accession No. XM-032424,FIG. 18)) (Data not shown). Southern blot hybridization with a clonedABCG2 probe confirmed the amplification of the correct cDNA (FIG. 19A).

RNA was isolated from cultured NIPs as well as from sorted SP cells andnon-SP controls (5000 and 10,000 cells respectively) using Trizol(Gibco) following the manufacturers protocol. Single stranded cDNA wasmade with the Superscript First-Strand System (Invitrogen, Carlsbad,Calif.). The cDNAs were amplified by polymerase chain reaction (PCR).Controls without reverse transcriptase (-RT controls) were done for allPCR reactions. PCR products were analyzed by agarose gel electrophoresisand the correct identity of products was confirmed by sequencing.Template concentrations were normalized for GAPDH (31 cycles). ABCG2,MDR1 and nestin were amplified with 34, 38, and 36 cycles respectively.Primers were: 5′tgaaggtcggagtcaacggatttggt3′ and5′catgtgggccatgaggtccaccac3′ for GAPDH, 5′agaggggaattcctggag3′ and5′ctgaggaccaggactctcta3′ for nestin, 5′tcctggagcggttctacgac3′ and5′gggcttcttggacaaccttttca3′ for MDR1 and 5 gctggggttctcttcttcctgacg3′and 5′ctaccccagccagtgtcaac3′ for ABCG2. PCR products for ABCG2 weretransferred to nylon membranes (Hybond N+; Amersham Pharmacia; LittleChalfont; GB). Blots were hybridized with radiolabeled, cloned ABCG2 orMDR1 probes using Rapid Hyb Buffer (Amersham) following themanufacturers protocol. Primers for the full open reading frame of humanABCG2 were 5′tattaagctgaaaagataaaaactctcc3′ and5′atgtgaggataaatcatactgaat3′ (base pairs 174-202 and 2184-2207 ofGenbank sequence XM_(—)032424).

EXAMPLE 8

1.5 to 2% of Cultured Cells Have a Side Population Phenotype in theHoechst 33342 Dye Exclusion Assay.

NIPs, linked to neural stem cells by their expression of nestin, alsoshow characteristics of bone marrow SP stem cells, by virtue of theirexpression of ABCG2 in a substantial subpopulation of NIPs identified bythe Hoechst 33342 exclusion assay. This side population of NIPs alsoexpresses MDR-1, contributing to their continued expansion in vitro.

To investigate whether the expression of ABCG2 lends a side population(SP) phenotype to some of the cultured NIPs, Hoechst 33342 dye exclusionassays were performed following the published protocol (Goodell et al.,1996, supra) with slight modifications. The exclusion of the Hoechst33342 dye, which defines the pluripotential side population (SP) ofhematopoietic stem cells, is mediated by the ATP-binding cassettetransporter, ABCG2. Verapamil was used as a specific inhibitor of H33342transport (Goodell et al., 1996, supra).

The Hoechst dye exclusion assay was done following the protocol ofGoodell et al. 1996, supra, with the modification that staining wasperformed while the cells were still attached to the culture dish. Cellviability with this change in protocol was significantly higher comparedto the original protocol in which cells are stained while in suspension.Hoechst 33342 (Sigma) was added to the growth media at a finalconcentration of 5 μg/ml and cells were incubated for 90 minutes at 37°C. Where indicated, verapamil (Sigma) was added 15 minutes prior to thestart of the assay at a final concentration of 50 μmol/l. For analysiscells were trypsinized, poured through a 40 μm cell strainer, andresuspended in ice cold Hanks' balanced salt solution containingpropidium iodide (Sigma) at a final concentration of 2 μg/ml for thediscrimination of dead cells. Flow cytometry was done on a Vantage and aMoflow cell sorter using a UV laser for excitation of Hoechst 33342 andpropidium iodide. 450/20 BP (blue) and 630/20 (red) filters were usedfor analysis in linear mode.

Fluorescence activated cell sorting (FACS) showed a clearly visible sidepopulation (FIG. 19B; 2.1% gated cells) which was absent in the presenceof the inhibitor verapamil (FIG. 19C; 0.1% gated cells). In analyses oftwo other NIP cultures independently derived, the percentage ofSP-positive cells was 1.5 and 2 percent, respectively (data not shown).We also amplified the full open reading frame of ABCG2 from NIPs.Cloning into an expression vector and transfection into INS-1 insulinomacells resulted in effective Hoechst 33342 exclusion in transfected cells(data not shown).

EXAMPLE 9

The SP Phenotype Correlates with the Expression of ABCG2, MDR1, andNestin

SP cells and non-SP control cells were isolated from cultured NIPs byFACS (FIG. 20A; gates R1 and R2 respectively). Expression of ABCG2correlated well with the SP phenotype as shown by RT-PCR (FIG. 20B) andSouthern blot hybridization with a cloned ABCG2 probe. RT-PCR andSouthern blot hybridization demonstrated expression of MDR1((P-glycoprotein), another ATP-binding cassette transporter present inhematopoietic stem cells (Chaudhary et al., 1991, Cell 66:85)) in theSP-fraction of cultured NIPs (FIG. 20B). The SP fraction also expressednestin at a markedly higher level than the non-SP controls (FIG. 20B).

Nestin-positive cells derived from human pancreatic islets contain 1.5to 2% of SP cells, which express ABCG2 and nestin at high levelscompared to non-SP control cells. The correlation of ABCG2 expressionwith the SP phenotype confirms the finding that ABCG2 activity is amajor component of the SP dye efflux (Kim et al., supra; Scharenberg etal., supra; Zhou et al., supra). The coexpression of nestin and ABCG2indicates a broader role for nestin as a general marker ofstem/progenitor cells.

The SP cell fraction of the bone marrow in different mammalian speciesincluding humans contains the vast majority of multipotentialhematopoietic stem cells (HSCs) (Goodell et al., 1997, supra). Thedifferentiation of bone marrow-derived SP cells is not limited to bloodlineages; in animal models SP cells also differentiate to skeletal andcardiac muscle as well as endothelial cells (Gussoni et al., Nature401:390; Jackson et al., 2001, J Clin Invest 107:1395). In addition,bone marrow-derived stem cells differentiate into functional brain andliver cells (Brazelton et al., 2000, Science 290:1775; Lagasse et al.,2000, Nat Med 6:1229).

The portion of SP cells in the NIP cultures (1.5 to 2%) is at least20-fold higher than that found in the bone marrow (0.05%) (Goodell etal., 1996, supra). However, a comparable percentage of SP cells is foundin cultures of muscle satellite cells, which are considered to bemyogenic progenitors (Jackson et al., 1999, Proc Natl Acad Sci96:14482). In addition to generating differentiated myofibres muscle SPcells have been shown to reconstitute the bone marrow of lethallyirradiated mice, thus exhibiting an unexpected degree of plasticity(Gussoni et al., 1999, supra). Some evidence exists that these SP cellsalso come from the bone marrow before taking up residence in the muscle(Kawada et al., 2001, Blood 98:2008). An SP population has also beendemonstrated in mouse embryonic stem cells (Zhou et al., 2001, supra).The expression on ABCG2 neural stem cells has been shown to besignificantly higher than it is in more differentiated neuralcells(Geschwind et al., 2001, Neuron 29:325).

MDR1 (P-glycoprotein, ABCB1) is another ATP-binding cassette transporter-expressed in hematopoietic stem cells (among several other cell types)(Zhou et al., 2001, supra; Chaudhary et al., 1991, supra).Overexpression of MDR1 in bone marrow cells leads to an expansion of theSP population, their prolonged survival in culture and an enhancedrepopulating activity after transplantation into mice (Bunting et al.,2000, supra). In some of the transplanted animals it ultimately resultsin a myeloproliferative syndrome resembling chronic myelogenous leukemia(Bunting et al., 1998, Blood 92:2269). Therefore MDR-1 expression hasbeen implicated in the expansion of hematopoietic stem cells and wassuggested as a characteristic of proliferating stem cells in contrast toquiescent cells, only expressing ABCG2 (Bunting et al., 2002, supra).MDR1 is expressed in the SP fraction of pancreatic islet derived NIPs.

EXAMPLE 10

Immunocytochemical Identification of GLP-1R-positive Human PancreaticStem Cells

Nestin-positive pancreatic islet stem cells were analyzed for GLP-1Rexpression. Human islet tissue was obtained from the Juvenile DiabetesResearch Center for Islet Transplantation, Harvard Medical School. NIPswere isolated as described previously (Zulewski, et al., 2001, Diabetes50: 521). Briefly, islets were washed and cultured in RPMI 1640 mediumcontaining serum, 11.1 mM glucose, antibiotics, sodium pyruvate,β-mercaptoethanol, and growth factors. Within several days,nestin-positive cells (identified immunocytochemically as describedabove) grew out from the islets. Later, these cells were cloned andexpanded in medium containing 20 ng/ml of basic fibroblast growth factorand epidermal growth factor or 1000 units of recombinant human leukemiainhibitory factor. Incubation with GLP-1 was administered in the absenceof serum and fresh peptide was added every 48 h without changing themedium.

Immunocytochemical detection of GLP-1R was performed using rabbitpolyclonal antisera (Heller et al., supra) as follows. Cells cultured onLab-Tek chamber slides (Nunc, Naperville, Ill.) or gridded coverslips(Bellco Glass, NJ) were fixed with 4% paraformaldehyde in PBS for 10minutes at room temperature. After several rinses in PBS, cells wereblocked with normal donkey serum for 30 minutes and incubated withprimary antisera or pre-immune sera at 4° C. The following day, cellswere rinsed with PBS and incubated with secondary antisera (donkeyanti-rabbit and donkey anti-guinea pig) labeled with Cy-3 or Cy-2 for 1hour at room temperature. After several washes, coverslips containingcells were mounted onto slides in mounting medium containing DAPI(Vector Laboratories, Burlingame, Calif.) which stains nuclei.Fluorescence images were obtained using a Zeiss epifluorescencemicroscope equipped with an Optronics TEC-470 CCD camera (OptronicsEngineering, Goleta, Calif.) interfaced with a Powermac 7100. IP labSpectrum software (Signal Analytics, Vienna, Va.) was used to acquireand analyze images.

GLP-1R immunoreactivity was detected in the majority of NIPs (at least60%) examined (FIG. 22A).

EXAMPLE 11

Identification of GLP-R1-positive Human Stem Cells by RT-PCR

To confirm the immunocytochemical identification of GLP-1R expression inpancreatic islets (NIPs), RT-PCR was performed using of total RNAprepared from NIPs. RT-PCR was performed as described above in example6, for the identification of nestin mRNA with the difference being thatthe following GLP-1R-specific primers were used: 5′ gtgtggcggccaattactac3′ (Forward); 5′ cttggcaagtctgcatttga 3′ (Reverse). Amplification of NIPmRNA produced the expected 346 bp product (FIG. 22B) indicating that, inaddition to expressing the GLP-1R protein, NIPs have the biosyntheticability to produce GLP-1R. GLP-1R, therefore, in addition to nestin, isuseful in the present invention as a marker for pancreatic stem cells.

EXAMPLE 12

In Vitro Proliferation of Nestin Positive Stem Cells

The ability of nestin-positive stem cells to proliferate in vitro wasdetermined.

Islets prepared from 60 day-old rats or a normal adult human were firstplated on concanavalin-A-coated dishes and cultured in modified RPMI1640 medium containing 10% fetal bovine serum for four days to purge theislet preparation of fibroblasts and other non-islet cells that adheredto the ConA-coated plates. The islets that did not adhere to the platesunder these culture conditions were collected and transferred to 12-wellplates (without ConA coating) containing the same modified RPMI 1640medium now additionally supplemented with bFGF and EGF (20 ng/mL each).The growth factors bFGF and EGF together were selected because they areknown to stimulate the proliferation of neural stem cells derived fromependyma of the brain (Reynolds and Weiss, 1996, Dev. Biol., 175:1-13).The islets attached to the plates and cells slowly grew out of the isletas a monolayer (estimated cell doubling time 40-45 hrs in human cells).The outgrowing monolayer of cells were phenotypically homogenous (FIG.9A, panel 1) and expressed nestin (FIG. 9A, panel 2). Rat cells werepicked from the monolayer (batches of at least 20-30 cells), subclonedinto 12-well plates, and incubated with the modified RPMI 1640 medium(11.1 mM glucose) containing bFGF and EGF. The subcloned cells grewrapidly and became confluent at six days with an estimated cell doublingtime of 12-15 hrs (FIG. 9A, panel 3), and by 12 days formed wave-likestructures. After 15-17 days of culture, the cells formed islet-likeclusters (ILCs) (FIG. 9A, panel 4). Similar cells were cloned from humanislets (FIG. 9B). Upon reaching confluence (FIG. 9B, panel 1), the humancells migrated to form large vacuolated structures in the dish (FIG. 9B,panels 2 and 3). The cells lining the large spaces then changedmorphology, rounded, and aggregated together forming three dimensionalILCs (FIG. 9B, panels 4-6).

Indicators of differentiation of these nestin-positive islet progenitorcells (NIPs) that formed these ILCs were characterized by RT-PCR andSouthern blot and found that they express the endocrine marker NCAM(neural cell adhesion molecule) (Cirulli et al., 1994, J. Cell Sci.,107:1429-36) (FIG. 9C, right panel) and the ductal cell marker CK19(cytokeratin 19) (Bouwens et al., 1998, J. Pathol., 184:234-9; Bouwenset al., 1995, J. Histochem. Cytochem., 43:245-53; Bouwens et al., 1994,Diabetes, 43:1279-93) (FIG. 9C, left panels). At this stage of thestudies it was concluded that when the NIPs became confluent andaggregated into islet-like cell clusters, they began to expresspancreatic genes (NCAM and CK19), but were limited in expression ofislet genes because of the absence of growth factors essential for theirdifferentiation to endocrine cells. It was also recognized that thedifferentiation of a progenitor cell population typically requires firsta proliferative phase and then quiescence of proliferation in thepresence of differentiation-specific morphogen growth factors. Thereforethe culture conditions were modified in some instances by replacing themedia containing 11.1 mM glucose, bFGF and EGF, which inducesproliferation of cells, with media containing lower glucose (2.5 mM),which is less proliferative, and the factors HGF/Scatter Factor orbetacellulin and Activin A. Glucose is a known proliferative factor forpancreatic islet β-cells (Swenne, 1992, Diabetologia, 35:193-201;Bonner-Weir, 1989, Diabetes, 38:49-53) and both HGF/Scatter Factor andActivin A have been shown to differentiate the pancreatic ductal cellline AR42J into an endocrine phenotype that produces insulin, glucagon,and other pancreatic endocrine cell proteins (Mashima et al., 1996,Endocrinology, 137:3969-76; Mashima et al., 1996, J. Clin. Invest.,97:1647-54).

Cultures containing ILCs expressed the pancreas-specific homeodomainprotein IDX-1 by immunocytochemistry (FIG. 10A, upper panel), RT-PCR andSouthern blot (FIG. 10B), and by Western immunoblot (FIG. 10C). The ILCsalso expressed the mRNA encoding proglucagon as seen by RT-PCR (FIG.10D) and produced immunoreactive glucagon, glucagon-like peptide-1, andinsulin. Radioimmunoassays of media obtained following 72-96 h ofculture of islet-like clusters in several wells gave values of 40-80pg/ml GLP-1, 30-70 pg/ml glucagon, 29-44 pg/ml insulin.Radioimmunoassays were performed as follows.

Insulin and glucagon concentrations in culture media were determined byultra sensitive radioimmunoassay kits purchased from Linco Research Inc.and DPC Inc., respectively. The antisera supplied in the respective kitsare guinea pig anti-human insulin and rabbit anti-human glucagon. GLP-1secretion was measured with an anti-human GLP-1(7-36)amide rabbitpolyclonal antiserum raised by immunization of a rabbit with a syntheticpeptide CFIAWLVKGR amide conjugated to keyhole limpet hemocyanin. Theantiserum is highly specific for the detection of GLP-1(7-36)amide andonly weakly detects proglucagon. The sensitivity levels for these assaysare 6 pg/mL, 13 pg/mL and 10.2 pg/mL, respectively.

Incubation of the ILCs for 7 days in 10 mM nicotinamide, as described byRamiya et al. (Ramiya et al., 2000, Nat. Med., 6:278-282), increasedinsulin secretion by 2- to 3-fold.

Several additional pancreatic markers were expressed in differentiatedNIPs such as glucose transporter-2 (Wang et al., 1998), synaptophysin,and HGF (Menke et al., 1999) as shown in FIG. 15. To determine whetherthe differentiating NIPs may have properties of pancreatic exocrinetissue, we used RT-PCR and detected the expression of amylase andprocarboxypeptidase (FIG. 15).

Some cultures of NIPs containing stem cells also expressed the mRNAencoding proglucagon and insulin as seen by RT-PCR (FIG. 16A and B).

The expression of IDX-1 is of particular importance because it isrecognized to be a master regulator of pancreas development, andparticularly to be required for the maturation and functions of thepancreatic islet β-cells that produce insulin (Stoffers et al., 1997,Trends Endocrinol. Metab., 8:145-151).

Because the neogenesis of new islets is also known to occur bydifferentiation of cells in pancreatic ducts, particularly during theneonatal period (rats and mice) but to some extent throughout adult life(Bonner-weir et al., 1993, Diabetes, 42:1715-1720; Rosenberg, 1995, CellTransplant, 4:371-383; Bouwens et al., 1996, Virchows Arch.,427:553-560), nestin expression was analyzed in the pancreatic ducts ofadult rats. By dual fluorescence immunocytochemistry with antisera tonestin and to cytokeratin 19, a marker of ductal epithelium, nestin isstrongly expressed in localized regions of both the large and smallducts, as well as in some centrolobular ducts within the exocrine acinartissue (FIGS. 11A and 11B). Remarkably, the localized regions of nestinexpression in the ducts are mostly devoid of staining with the anti CK19antiserum. Further, the nestin-positive cells in the ducts appear tohave a morphology that is distinct from that of the epithelial cells.The epithelial cells consist of a homogenous population of cuboidal,rounded cells, whereas the nestin-positive cells are nucleated,serpiginous and appear to reside in the interstices among or aroundepithelial cells (FIG. 11C).

Thus, CK19 is not expressed in the majority of ductal cells that expressnestin suggesting that these nestin-expressing cells located within thepancreatic ducts are a passenger population of cells distinct from theductal epithelial cells and are stem cells that have not yetdifferentiated into a ductal or endocrine phenotype. The finding oflocalized populations of nestin-expressing cells within the pancreaticducts and islets of the adult rat pancreas further supports the ideathat rat pancreatic ducts contain cells that are progenitors of isletcells (neogenesis), but these progenitors are not a subpopulation ofductal epithelial cells per se.

EXAMPLE 13

Transplantation of Pancreatic Stem Cells Engineered to Express IDX-1 inHuman Subjects with Diabetes Mellitus

Islets isolated from pig or human donor pancreata, or from pancreaticbiopsy of eventual human transplant recipient are cultured ex vivo inconditions that stimulate outgrowth of stem cells. Stem cells are thenisolated away from islets (cloned), expanded in vitro in proliferationmedia containing bFGF-2, EGF, and 11.1 mM glucose, transfected/injectedwith an expression vector containing DNA encoding transcriptionfactor-IDX-1, and transplanted into a diabetic recipient. Alternatively,IDX-1-transfected stem cells are treated with GLP-1, or otherdifferentiation morphogens or growth factors for 1-3 days beforetransplantation to initiate processes of differentiation of engineeredstem cells to β-cells. In one embodiment, stem cells are neitherexpanded or differentiated prior to administration to the recipient orare only expanded or differentiated prior to administration to therecipient. In one embodiment, GLP-1 is administered to the recipientduring and for several days after transplantation to stimulatedifferentiation of stem cells and encourage successful engraftment.According to this method, xenographs (pig islets) or allographs (humanislets from a human donor that is not the recipient), as well asisographs (islets derived from the recipient) are carried out. It ishypothesized that when transplanted to a host recipient the stem cellgenetic repertoire is reprogrammed so that the host recognizes the stemcells (in the case of xenographs or allographs) as self, such thatimmune intolerance and graft rejection and destruction by autoimmunity(type 1 diabetes) does not occur.

EXAMPLE 14

Transplantation of Pancreatic Stem Cells Cultured to StimulateExpression of IDX-1 in Human Subjects with Diabetes Mellitus

Islets isolated as described are cultured ex vivo for several days inconditions that stimulate first the expansion (proliferation) of stemcells that exist within the islets and then the expression oftranscription factor HDX-1. The proliferation of stem cells is achievedby culturing the islets in media containing bFGF-2, EGF, and 11.1 mMglucose. Induction of the expression of IDX-1 is achieved by incubationin the presence of GLP-1 and 2 mM glucose. The islets so preconditionedby the treatments described are transplanted to the host recipient.Additionally, the host recipient may be administered GLP-1 during andfor several days after the transplantation to further expand anddifferentiate stem cells to insulin-producing cells to enhance successof engraftment.

According to this method, xenographs (pig islets), allographs (humanislets from a human donor that is not the recipient), as well asisographs (islets derived from the recipient) are effectuated.

EXAMPLE 15

Xenogeneic Transplantation of Pancreatic Stem Cells into the Kidney

Human nestin-positive-islet progenitor cells (NIPS) were isolated asdescribed, and transplanted under the renal capsules of eight C57B16mice that were not immunosuppressed. The transplanted human cells werenot rejected by the mouse recipient. Current understanding is that axenograft, such as human tissue, would be rejected by the mouse within5-10 days. Contrary to current understanding, we found that in 8 of the8 non-immunosuppressed mice tested to date, all of the transplantssuccessfully engrafted and proliferated into large masses of tissueengulfing the pole of the kidney by one month (30-38 days) after atransplantation of approximately 10⁵ to 10⁶ cells.

One C57B16 mouse was sacrificed and determined to have a large area ofnew growth at the site of transplantation. A section of the kidney thatincluded the new tissue was divided into two pieces; one piece wasfrozen for frozen section histology, and the other piece was fixed inparaformaldehyde for paraffin section histology. Frozen sections wereprepared and stained with hematoxylin and eosin (H&E) and antisera tovarious islet cell antigens.

Examination of the H&E stained kidney section demonstrated the presenceof a new growth that was not part of the kidney, exhibiting apleiomorphic morphology consisting of a mixed mesenchymal and epithelialtissue containing hepatic, neural, ductal, adipodipic and hematopoeticcomponents. Photomicrographs of the kidney section demonstrated that thenew growth seemed to be invading the renal parenchyme, and theglomeruli. Specific immunostaining with human-specific (not mouse)antisera revealed cords of immunopositive cells staining forhuman-specific keratins, vimentin, and the CD45 leukocyte antigenspecific for human hematapoetic lymphocytes. The kidney of a secondC57B16 mouse also had a similar looking new growth at the site of theNIP transplantation.

The paraffin section of the NIP-engrafted kidney of a C57B16 mouse (thefirst mouse to be sacrificed) was examined. The tissue block that wasexamined was from the top of the kidney and showed the foreign tissue tobe well contained under the renal capsule with no signs of “invasion”into the renal parenchyma. Notably, amongst the pleiomorphic-lookinggraft tissues were areas that resembled renal parenchyma. Without beingbound to theory one hypothesis is that the graft consists of stem cellstrying to differentiate and that the stem cells are not “invading” butsimply migrating and proliferating and looking for a niche, i.e.mesenchymal instructions. They may be receiving cues from the kidney andmay be attempting to differentiate into kidney. The graft cells may notbe malignant, but may be just stem cells attempting to carry out theirfunction.

EXAMPLE 16

Xenogeneic Transplantation of Pancreatic Stem Cells into the Pancreas

Human nestin-positive-islet progenitor cells (NIPS) are isolated asdescribed, and transplanted into the pancreas of mice that are notimmunosuppressed and are (a) injured by streptozotocin (to producestreptozotocin induced diabetes) treatment or (b) NOD mice in whichthere is an ongoing islet is with inflammation.

The pancreas of the transplanted animals is examined to determine if theNIPs find their proper niche, receive instructions from the isletregion, and differentiate into islet (β-cell) cells.

EXAMPLE 17

Treatment of Diabetes by Xenogeneic Transplantation of Pancreatic StemCells

Human islets are isolated as described and cultured for several days invitro to expand the stem cell population. Human NIPS are transplanted tothe liver via the portal vein (according to conventional procedures wellknown in the art for transplantation to the liver.

Alternatively, a population of human NIPs (isolated as described) areintroduced into the blood stream. In certain embodiments, the human NIPSare introduced via the pancreatic artery, to direct them to the diabeticpancreas.

A population of control (untransplanted animals) and transplantedanimals are analyzed for amelioration of the symptoms of diabetes (e.g.blood glucose levels, insulin levels, number of pancreatic β-cells.

EXAMPLE 18

Identification of Nestin Positive Stem Cells in the Liver

Rat livers were isolated and frozen section were prepared according tomethods known in the art and described herein.

Frozen sections of rat liver (6 μM) were immunostained with a rabbitpolyclonal anti-nestin serum. The immunofluorescent signal was developedby reaction of anti-donkey IgG serum tagged with the fluorophore, Cy3(yellow-orange color. Nestin-positive cells surrounding a possible largebiliary duct are depicted in FIG. 13A. Clusters of nestin positive cellssurrounding several small biliary ducts are depicted in FIG. 13B.

EXAMPLE 19

Differentiation of NIPs Toward Hepatic Phenote

Because of the reported apparent commonalties between hepatic stem cells(oval cells), hepatic stellate cells, and progenitor cells in thepancreas, and the observations that following some injuries, theregenerating pancreas undergoes liver metaplasia (Slack, 1995; Reddy etal., 1991; Bisgaard et al., 1991; Rao et al., 1996), we performed RT-PCRto detect liver-expressed genes in the stem cells. PCR products wereobtained for XBP-1, a transcription factor required for hepatocytedevelopment (Reimold et al., 2000), and transthyretin, a liver acutephase protein. Several other liver markers were also expressed such asα-fetoprotein (Dabeva et al., 2000), E-Cadherin (Stamatoglou et al.,1992), c-MET (Ikeda et al., 1998), HGF (Skrtic et al., 1999) andsynaptophysin (Wang et al., 1998); see FIG. 15)) The expression ofproteins shared by the pancreas and liver, such as HGF andsynaptophysin, may reflect their common origin from the embryonicforegut endoderm, and represent differentiation toward either pancreaticor hepatic phenotypes.

EXAMPLE 20

GLP-1R Signalling in NIPs

The application of GLP-1 amide to single, isolated NWPs elevates levelsof intracellular Ca²⁺ concentration ([Ca²⁺]_(i)). Cells were plated ontogridded coverslips to permit subsequent immunohistochemical staining ofthe same cells to test for nestin expression. All cells from which[Ca²⁺]_(i) recordings were made were shown to express nestin. Incontrast to adult β-cells, GLP-1 stimulates [Ca²⁺]_(i) levels at basal(5.6 mM) glucose but was ineffective in the presence of high (20 mM)glucose (FIG. 23, panel A). These effects were reproduced by forskolin(FIG. 23, panel B) suggesting that the effects of GLP-1 are mediatedthrough the activation of Gs and cAMP production, the same signallingpathway used in normal β-cells. The pretreatment of single isolated NIPswith the peptide Extendin (9-39), a specific antagonist of GLP-1,prevents the increase in [Ca²⁺]_(i) mediated by GLP-1 (FIG. 23, panels Cand D). The effects of GLP-1R antagonist Extendin (9-39) suggests thatthe same isoform of GLP-1R is expressed in NIPs as in β-cells. The[Ca²⁺]_(i) increase mediated by GLP-1 was inhibited by exprecellularLa³⁺ (5 μM) suggesting that GLP-1 is activating [Ca²⁺]_(i) influx,consistent with its known role to depolarize β-cells (FIG. 23, panel E).We further demonstrate that tolbutamide (100 μM) stimulates [Ca²⁺]_(i)elevation in NIPs indicating that they express ATP-sensitive K⁺ channels(FIG. 23, panel F). These data are consistent with GLP-1 inducedmembrane depolarization and activation of voltage-dependent Ca²⁺channels in NIPs, consistent with its mechanism of action in β-cells.

EXAMPLE 21

GLP-1 Induces Differentiation of NIPs into Insulin-secreting Cells

Previous studies have demonstrated the insulinotropic action of GLP-1 aswell as its ability to stimulate β-cell neogenesis in partialpancreactectomized rats (Xu et al., 1999, Diabetes 48:2270). NIPs werepicked from islet cultures and expanded in growth medium containing bFGFplus EGF (passage 01) for 3-10 days (Zulewski et al., 2001 Diabetes 50:521). In certain instances NIPOs that were expanded for 3-5 days werefixed and subjected to immunocytochemical detection of Nestin (Cy-3) andInsulin (Cy-2) as shown in FIG. 24A. It was found that at this stagethey are nestin positive and insulin negative. When NIP cultures wereexpanded for 7-10 days and then treated with either GLP-1 or its stableanalog extendin-4, a subset of cells became insulin positive (Cy-2; FIG.24B, panel 3, 5, and 6) and Idx-1 positive (Cy-3; FIG. 24B, panel 7) andnestin negative (Cy-3; FIG. 23B, panel 4). There was an overall decreasein nestin expression when cultures were treated with GLP-1 (FIG. 24B,panel 2 vs. 4). Accordingly, treatment with Extendin-4 induced a 2- to3-fold increase in insulin secretion (FIG. 25). However, in some culturewells, confluence alone was sufficient to initiate small amounts ofinsulin secretion.

The homeodomain protein Idx-1 is critical for pancreas development andplays a major role in the transcriptional regulation of the insulingene. Haploindufficiency of Idx-1 expression results in a form of earlyonset type-2 diabetes (MODY4) and inherited amino acid changes in Idx-1are associated with late onset type-2 diabetes. Idx-1 is expressed indifferentiated NIP cell populations.

Human NIPs that had been repeatedly passaged lose their ability tosecrete insuling in response to GLP-1. However, transfection of thesecells with an expression vector encoding the human Idx-1 cDNA renderedthem responsive to GLP-1 and induced the synthesis of insulin in asubset of NIPs (FIG. 26, panel 1). Interestingly, transfection of NIPs(passage 9) with Idx-1 in the absence of GLP-1 treatment did not induceinsulin biosynthesis (FIG. 26, lower panel B), but did stimulate Idx-1expression levels (FIG. 26, upper panel B). Taken together, theseresults suggest that GLP-1 stimulates levels of Idx-1 expression in NIPcells and that a critical threshold concentration of Idx-1 is requiredfor NIPs to convert into insulin-producing cells, thus suggesting a rolefor the GLP-1R in islet cell differentiation.

REFERENCES

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OTHER EMBODIMENTS

Other Embodiments are within the claims that follow.

1. A method of isolating a stem cell from a pancreatic islet ofLangerhans, comprising the steps of: (a) removing a pancreatic isletfrom a donor; (b) culturing cells from the pancreatic islet underconditions wherein said cultured cells comprise nestin-positive cellswhich have migrated from said islet; and (c) selecting saidnestin-positive cells from the culture.
 2. The method of claim 1,wherein said nestin-positive clone is also an ABCG2-positive clone. 3.The method of claim 1, wherein said nestin-positive clone is alsopositive for at least one of the markers selected from the groupconsisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1,Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.
 4. Themethod of claim 1, wherein said nestin-positive clone does not expressat least one of the markers selected from the group consisting of CD34,CD45, CD133, MHC class I and MHC class II.
 5. The method of claim 1,wherein said migrated cells from step b form a monolayer.
 6. A method ofisolating a stem cell from a pancreatic islet of Langerhans, comprisingthe steps of: (a) removing a pancreatic islet from a donor; (b)culturing cells from the pancreatic islet under conditions wherein saidcultured cells comprise ABCG2 positive cells which have migrated fromsaid islet; and (c) selecting said ABCG2-positive cells from theculture.
 7. The method of claim 1 or 6, wherein the culturing is firstperformed in a vessel coated with concanavalin A and then againperformed in a vessel not coated with concanavalin A.
 8. The method ofclaim 1 or 6, comprising the additional step of: (d) expanding thenestin-positive cells or the ABCG2-positive cells by treatment with anagent selected from the group consisting of EGF, bFGF-2, high glucose,KGF, HGF/SF, GLP-1, exendin-4, IDX-1, a nucleic acid molecule encodingIDX-1, betacellulin, activin A, TGF-β, and combinations thereof.
 9. Themethod of claim 6 comprising the additional step of: (d) expanding theABCG2-positive clone by treatment with an agent selected from the groupconsisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4,IDX-1, a nucleic acid molecule encoding IDX-1, betacellulin, activin A,TGF-β, and combinations thereof.
 10. The method of claim 6, wherein saidmigrated cells from step b form a monolayer
 11. A method of inducing thedifferentiation of an isolated nestin-positive pancreatic stem cell intoa pancreatic progenitor cell, comprising the step of: treating anestin-positive pancreatic stem cell with an agent selected from thegroup consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, IDX-1, anucleic acid molecule encoding IDX-1, GLP-1, exendin-4, betacellulin,activin A, TGF-β, and combinations thereof, whereby the stem cellsubsequently differentiates into a pancreatic progenitor cell.
 12. Themethod of claim 11, wherein said nestin-positive pancreatic stem cell isalso an ABCG2-positive clone.
 13. The method of claim 11, wherein saidnestin-positive pancreatic stem cell is also positive for at least oneof the markers selected from the group consisting of Oct3/4, GLP-1receptor, latrophilin (type 2), Hes-1, [Nestin], Integrin subunits α6and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.
 14. The method of claim 11,wherein said nestin-positive pancreatic stem cell does not express atleast one of the markers selected from the group consisting of CD34,CD45, CD133, MHC class I and MHC class II.
 15. A method of inducing thedifferentiation of an isolated ABCG2-positive pancreatic stem cell intoa pancreatic progenitor cell, comprising the step of: treating anABCG2-positive pancreatic stem cell with an agent selected from thegroup consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, IDX-1, anucleic acid molecule encoding IDX-1, GLP-1, exendin-4, betacellulin,activin A, TGF-β, and combinations thereof, whereby the stem cellsubsequently differentiates into a pancreatic progenitor cell.
 16. Themethod of claim 11 or 15, wherein the pancreatic progenitor cellsubsequently forms pseudo-islet like aggregates.
 17. An isolated,nestin-positive human pancreatic or liver stem cell that is not a neuralstem cell.
 18. The isolated stem cell of claim 17, wherein said cell isalso ABCG2-positive.
 19. The isolated stem cell of claim 17, whereinsaid cell is also positive for at least one of the markers selected fromthe group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2),Hes-1, [Nestin], Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, orKir 6.2.
 20. The isolated stem cell of claim 17, wherein said cell doesnot express at least one of the markers selected from the groupconsisting of CD34, CD45, CD133, MHC class I and MHC class II.
 21. Anisolated, ABCG2-positive human pancreatic or liver stem cell that is nota neural stem cell.
 22. The isolated cell of claim 21, wherein said cellis also positive for at least one of the markers selected from the groupconsisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1,Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.23. The isolated cell of claim 21, wherein said cell does not express atleast one of the markers selected from the group consisting of CD34,CD45, CD133, MHC class I and MHC class II.
 24. The isolated stem cell ofclaim 21, wherein said cell does not express at least one of the markersselected from the group consisting of CD34, CD45, CD133, MHC class I andMHC class II.
 25. The isolated stem cell of claim 17 or 21, thatdifferentiates to form insulin-producing beta cells.
 26. The isolatedstem cell of claim 17 or 21, that differentiates to formglucagon-producing alpha cells.
 27. The isolated stem cell of claim 17or 21, that differentiates to form pseudo-islet like aggregates.
 28. Theisolated stem cell of claim 17 or 21, that differentiates to formhepatocytes.
 29. A pharmaceutical composition comprising the isolatedstem cell of claim 17 admixed with a physiologically compatible carrier.30. A pharmaceutical composition comprising the isolated stem cell ofclaim 18 admixed with a physiologically compatible carrier.
 31. Apharmaceutical composition comprising the isolated stem cell of claim 21admixed with a physiologically compatible carrier.